Co-reporter:Sydney E. Morris, Aaron W. Feldman, and Floyd E. Romesberg
ACS Synthetic Biology October 20, 2017 Volume 6(Issue 10) pp:1834-1834
Publication Date(Web):June 27, 2017
DOI:10.1021/acssynbio.7b00115
To bestow cells with novel forms and functions, the goal of synthetic biology, we have developed the unnatural nucleoside triphosphates dNaMTP and dTPT3TP, which form an unnatural base pair (UBP) and expand the genetic alphabet. While the UBP may be retained in the DNA of a living cell, its retention is sequence-dependent. We now report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the UBP and its mispairs in a variety of sequence contexts. Correct UBP synthesis is as efficient as for a natural base pair, except in one sequence context, and in vitro performance is correlated with in vivo performance. The data elucidate the determinants of efficient UBP synthesis, show that the dNaM-dTPT3 UBP is the first generally recognized natural-like base pair, and importantly, demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts for the expansion of the genetic alphabet.Keywords: DNA; hydrophobic; unnatural base pair;
Co-reporter:Deepak Thirunavukarasu, Tingjian Chen, Zhixia Liu, Narupat Hongdilokkul, and Floyd E. Romesberg
Journal of the American Chemical Society March 1, 2017 Volume 139(Issue 8) pp:2892-2892
Publication Date(Web):February 20, 2017
DOI:10.1021/jacs.6b13132
RNA or single-stranded DNA aptamers with 2′-F pyrimidines have been pursued to increase resistance to nucleases, and while it seems likely that these and other modifications, including the modification of purines, could be used to optimize additional properties, this has been much less explored because such aptamers are challenging to discover. Using a thermostable DNA polymerase, SFM4-3, which was previously evolved to accept nucleotides with 2′-modifications, we now report the selection of 2′-F purine aptamers that bind human neutrophil elastase (HNE). Two aptamers were identified, 2fHNE-1 and 2fHNE-2, that bind HNE with reasonable affinity. Interestingly, the 2′-F substituents facilitate the selection of specific interactions with HNE and overcome nonspecific electrostatic interactions that can otherwise dominate. The data demonstrate that inclusion of only a few 2′-F substituents can optimize properties far beyond simple nuclease resistance and that SFM4-3 should prove valuable for the further exploration and production of aptamers with properties optimized for various applications.
Co-reporter:Tingjian Chen and Floyd E. Romesberg
Journal of the American Chemical Society July 26, 2017 Volume 139(Issue 29) pp:9949-9949
Publication Date(Web):July 17, 2017
DOI:10.1021/jacs.7b03981
There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2′-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2′-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.
Co-reporter:Ramkrishna Adhikary, Jörg Zimmermann, and Floyd E. Romesberg
Chemical Reviews February 8, 2017 Volume 117(Issue 3) pp:
Publication Date(Web):January 20, 2017
DOI:10.1021/acs.chemrev.6b00625
Vibrational spectroscopy provides a direct route to the physicochemical characterization of molecules. While both IR and Raman spectroscopy have been used for decades to provide detailed characterizations of small molecules, similar studies with proteins are largely precluded due to spectral congestion. However, the vibrational spectra of proteins do include a “transparent window”, between ∼1800 and ∼2500 cm–1, and progress is now being made to develop site-specifically incorporated carbon–deuterium (C–D), cyano (CN), thiocyanate (SCN), and azide (N3) “transparent window vibrational probes” that absorb within this window and report on their environment to facilitate the characterization of proteins with small molecule-like detail. This Review opens with a brief discussion of the advantages and limitations of conventional vibrational spectroscopy and then discusses the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study, including electrostatics, stability and folding, hydrogen bonding, protonation, solvation, dynamics, and interactions with inhibitors. The use of the probes to vibrationally image proteins and other biomolecules within cells is also discussed. We then present four case studies, focused on ketosteroid isomerase, the SH3 domain, dihydrofolate reductase, and cytochrome c, where the transparent window vibrational probes have already been used to elucidate important aspects of protein structure and function. The Review concludes by highlighting the current challenges and future potential of using transparent window vibrational probes to understand the evolution and function of proteins and other biomolecules.
Co-reporter:Aaron W. Feldman, Vivian T. Dien, and Floyd E. Romesberg
Journal of the American Chemical Society February 15, 2017 Volume 139(Issue 6) pp:2464-2464
Publication Date(Web):February 7, 2017
DOI:10.1021/jacs.6b12731
We have developed an unnatural base pair (UBP) and a semisynthetic organism (SSO) that imports the constituent unnatural nucleoside triphosphates and uses them to replicate DNA containing the UBP. However, propagation of the UBP is at least in part limited by the stability of the unnatural triphosphates, which are degraded by cellular and secreted phosphatases. To circumvent this problem, we now report the synthesis and evaluation of unnatural triphosphates with their β,γ-bridging oxygen replaced with a difluoromethylene moiety, yielding dNaMTPCF2 and dTPT3TPCF2. We find that although dNaMTPCF2 cannot support in vivo replication, likely due to poor polymerase recognition, dTPT3TPCF2 can, and moreover, its increased stability can contribute to increased UBP retention. The data demonstrate the promise of this chemical approach to SSO optimization, and suggest that other modifications should be sought that confer phosphatase resistance without interfering with polymerase recognition.
Co-reporter:Aaron W. Feldman and Floyd E. Romesberg
Journal of the American Chemical Society August 23, 2017 Volume 139(Issue 33) pp:11427-11427
Publication Date(Web):August 10, 2017
DOI:10.1021/jacs.7b03540
In an effort to expand the genetic alphabet and create semi-synthetic organisms (SSOs) that store and retrieve increased information, we have developed the unnatural base pairs (UBPs) dNaM and d5SICS or dTPT3 (dNaM-d5SICS and dNaM-dTPT3). The UBPs form based on hydrophobic and packing forces, as opposed to complementary hydrogen bonding, and while they are both retained within the in vivo environment of an Escherichia coli SSO, their development was based on structure–activity relationship (SAR) data generated in vitro. To address the likely possibility of different requirements of the in vivo environment, we screened 135 candidate UBPs for optimal performance in the SSO. Interestingly, we find that in vivo SARs differ from those collected in vitro, and most importantly, we identify four UBPs whose retention in the DNA of the SSO is higher than that of dNaM-dTPT3, which was previously the most promising UBP identified. The identification of these four UBPs further demonstrates that when optimized, hydrophobic and packing forces may be used to replace the complementary hydrogen bonding used by natural pairs and represents a significant advance in our continuing efforts to develop SSOs that store and retrieve more information than natural organisms.
Co-reporter:Dr. Tingjian Chen; Floyd E. Romesberg
Angewandte Chemie International Edition 2017 Volume 56(Issue 45) pp:14046-14051
Publication Date(Web):2017/11/06
DOI:10.1002/anie.201707367
AbstractThe ability to amplify DNA along with its unprecedented sequence control has led to its use for different applications, but all are limited by the properties available to natural nucleotides. We previously reported the evolution of polymerase SFM4-3, which better tolerates 2′-modified substrates. To explore the utility of SFM4-3, we now report the characterization of its recognition of substrates with 2′-azido, 2′-chloro, 2′-amino, or arabinose sugars. We find that SFM4-3 can efficiently synthesize polymers composed of these nucleotides, and most interestingly, that SFM4-3 can also PCR amplify these modified oligonucleotides. When combined with post-amplification modification, the latter allows for the exponential amplification of polymers that may be functionalized with desired moieties arrayed in a controlled fashion, the utility of which we demonstrate with extensive small molecule functionalization and the production and initial characterization of a novel DNA hydrogel.
Co-reporter:Zhixia Liu;Tingjian Chen
Chemical Science (2010-Present) 2017 vol. 8(Issue 12) pp:8179-8182
Publication Date(Web):2017/11/20
DOI:10.1039/C7SC03747C
RNA or DNA aptamers with 2′-OMe-modifications have been pursued to increase resistance to nucleases, but have been difficult to identify because the OMe groups ablate polymerase recognition. We recently reported evolution of the thermostable DNA polymerases SFM4-6 and SFM4-9, which enable the efficient “transcription” and “reverse transcription”, respectively, of 2′-OMe oligonucleotides. With these polymerases, we now report the first selection of fully 2′-OMe modified aptamers, specifically aptamers that bind human neutrophil elastase (HNE). Two aptamers, 2mHNE-1 and 2mHNE-2, were isolated after five rounds of selection, and four more, 2mHNE-3–6, after an additional five rounds that included selection pressure for binding in the presence of serum. All six aptamers bind with reasonable affinity, which requires the 2′-OMe substituents. Further characterization of one aptamer, 2mHNE-5, showed that unlike a previously reported natural anti-HNE aptamer, affinity for HNE is retained in the presence of high concentrations of salt or serum. The polymerases SFM4-6 and SFM4-9 should prove valuable for the production and further exploration of modified aptamers.
Co-reporter:Yorke Zhang;Anne Xiaozhou Zhou;Brian M. Lamb;Aaron W. Feldman;Lingjun Li;Thomas Lavergne
PNAS 2017 Volume 114 (Issue 6 ) pp:1317-1322
Publication Date(Web):2017-02-07
DOI:10.1073/pnas.1616443114
All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic
alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase
the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would
thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import
them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS
UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required
growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly
a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used
a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that
had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely
retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information
using a six-letter, three-base-pair alphabet.
Co-reporter:Tingjian Chen, Narupat Hongdilokkul, Zhixia Liu, Deepak Thirunavukarasu, Floyd E Romesberg
Current Opinion in Chemical Biology 2016 Volume 34() pp:80-87
Publication Date(Web):October 2016
DOI:10.1016/j.cbpa.2016.08.001
•Modified nucleotides stabilize and increase the potential functions of DNA and RNA.•Modified nucleotides allow DNA and RNA to adopt protein-like structures.•Hydrophobic and packing forces may be used to expand the potential of DNA and RNA.•Nucleotides with unnatural nucleobases may be used to develop unnatural base pairs.•It is now possible to synthesize and even replicate modified or unnatural DNA in cells.DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymatic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.
Co-reporter:Thomas Lavergne, Rajan Lamichhane, Denis A. Malyshev, Zhengtao Li, Lingjun Li, Edit Sperling, James R. Williamson, David P. Millar, and Floyd E. Romesberg
ACS Chemical Biology 2016 Volume 11(Issue 5) pp:1347
Publication Date(Web):March 4, 2016
DOI:10.1021/acschembio.5b00952
Ribosome assembly has been studied intensively using Förster resonance energy transfer (FRET) with fluorophore-labeled fragments of RNA produced by chemical synthesis. However, these studies are limited by the size of the accessible RNA fragments. We have developed a replicable unnatural base pair (UBP) formed between (d)5SICS and (d)MMO2 or (d)NaM, which efficiently directs the transcription of RNA containing unnatural nucleotides. We now report the synthesis and evaluation of several of the corresponding ribotriphosphates bearing linkers that enable the chemoselective attachment of different functionalities. We found that the RNA polymerase from T7 bacteriophage does not incorporate NaM derivatives but does efficiently incorporate 5SICSCO, whose linker enables functional group conjugation via Click chemistry, and when combined with the previously identified MMO2A, whose amine side chains permits conjugation via NHS coupling chemistry, enables site-specific double labeling of transcribed RNA. To study ribosome assembly, we transcribed RNA corresponding to a 243-nt fragment of the central domain of Thermus thermophilus 16S rRNA containing 5SICSCO and MMO2A at defined locations and then site-specifically attached the fluorophores Cy3 and Cy5. FRET was characterized using single-molecule total internal reflection fluorescence (smTIRF) microscopy in the presence of various combinations of added ribosomal proteins. We demonstrate that each of the fragment’s two three-helix junctions exist in open and closed states, with the latter favored by sequential protein binding. These results elucidate early and previously uncharacterized folding events underlying ribosome assembly and demonstrate the applicability of UBPs for biochemical, structural, and functional studies of RNAs.
Co-reporter:Ramkrishna Adhikary, Jörg Zimmermann, Philip E. Dawson, and Floyd E. Romesberg
Analytical Chemistry 2015 Volume 87(Issue 22) pp:11561
Publication Date(Web):November 2, 2015
DOI:10.1021/acs.analchem.5b03437
Cyano and thiocyano groups have received attention as IR probes of local protein electrostatics or solvation, due to their strong absorptions and the ability to site specifically incorporate them within proteins. However, interpreting their spectra requires knowing whether they engage in hydrogen bonds (H-bonds). Existing methods for the detection of such H-bonding interactions are based on structural analysis or correlations between IR and NMR signals and are labor intensive and possibly ambiguous. Here, using model systems we show that the absorption frequency of both probes is linearly correlated with temperature and that the slope of the resulting line (frequency–temperature line slope or FTLS) reflects the nature of the probe’s microenvironment, including whether or not the probe is engaged in H-bonds. We then show that the same linear dependence is observed with p-cyano phenylalanine, cyanylated cysteine, or cyanylated homocysteine incorporated at different positions within the N-terminal Src homology 3 domain of the murine adapter protein Crk-II. The FTLSs indicate that p-cyano phenylalanine incorporated at two positions is engaged in strong H-bonding, while it is involved in weaker H-bonding at a third position. In contrast, the FTLS of the cyanylated cysteine or cyanylated homocysteine absorptions indicates that they do not engage in H-bonding at either a buried or surface exposed position. While the differences likely reflect side chain flexibility and the probe’s ability to avoid solvent, the data suggest that the temperature dependence of the absorption provides a simple method to gauge the probe’s environment, including the presence of H-bonding.
Co-reporter:Ramkrishna Adhikary, Wayne Yu, Masayuki Oda, Ross C. Walker, Tingjian Chen, Robyn L. Stanfield, Ian A. Wilson, Jörg Zimmermann, and Floyd E. Romesberg
Biochemistry 2015 Volume 54(Issue 11) pp:2085-2093
Publication Date(Web):March 10, 2015
DOI:10.1021/bi501417q
While adaptive mutations can bestow new functions on proteins via the introduction or optimization of reactive centers, or other structural changes, a role for the optimization of protein dynamics also seems likely but has been more difficult to evaluate. Antibody (Ab) affinity maturation is an example of adaptive evolution wherein the adaptive mutations may be identified and Abs may be raised to specific targets that facilitate the characterization of protein dynamics. Here, we report the characterization of three affinity matured Abs that evolved from a common germline precursor to bind the chromophoric antigen (Ag), 8-methoxypyrene-1,3,6-trisulfonate (MPTS). In addition to characterizing the sequence, molecular recognition, and structure of each Ab, we characterized the dynamics of each complex by determining their mechanical response to an applied force via three-pulse photon echo peak shift (3PEPS) spectroscopy and deconvoluting the response into elastic, anelastic, and plastic components. We find that for one Ab, affinity maturation was accomplished via the introduction of a single functional group that mediates a direct contact with MPTS and results in a complex with little anelasticity or plasticity. In the other two cases, more mutations were introduced but none directly contact MPTS, and while their effects on structure are subtle, their effects on anelasticity and plasticity are significant, with the level of plasticity correlated with specificity, suggesting that the optimization of protein dynamics may have contributed to affinity maturation. A similar optimization of structure and dynamics may contribute to the evolution of other proteins.
Co-reporter:Dr. Denis A. Malyshev ; Floyd E. Romesberg
Angewandte Chemie International Edition 2015 Volume 54( Issue 41) pp:11930-11944
Publication Date(Web):
DOI:10.1002/anie.201502890
Abstract
All biological information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two base pairs. Long-standing efforts to develop two synthetic nucleotides that form a third, unnatural base pair (UBP) have recently yielded three promising candidates, one based on alternative hydrogen bonding, and two based on hydrophobic and packing forces. All three of these UBPs are replicated and transcribed with remarkable efficiency and fidelity, and the latter two thus demonstrate that hydrogen bonding is not unique in its ability to underlie the storage and retrieval of genetic information. This Review highlights these recent developments as well as the applications enabled by the UBPs, including the expansion of the evolution process to include new functionality and the creation of semi-synthetic life that stores increased information.
Co-reporter:Dr. Denis A. Malyshev ; Floyd E. Romesberg
Angewandte Chemie 2015 Volume 127( Issue 41) pp:12098-12113
Publication Date(Web):
DOI:10.1002/ange.201502890
Abstract
Seit dem letzten gemeinsamen Vorfahren allen Lebens auf der Erde wird die gesamte biologische Information durch ein genetisches Alphabet codiert, das aus nur vier Nucleotiden und den von ihnen gebildeten zwei Basenpaaren besteht. Aus langjährigen Versuchen, aus zwei synthetischen Nucleotiden ein drittes, nichtnatürliches Basenpaar (UBP) zu formen, haben sich jetzt drei vielversprechende Kandidaten herauskristallisiert. Eines dieser UBPs basiert auf alternativen Wasserstoffbrücken, während die beiden anderen durch hydrophobe Kräfte und Stapelkräfte stabilisiert werden. Alle drei dieser UBPs werden bemerkenswert effizient und zuverlässig repliziert und transkribiert. Die beiden letztgenannten UBP-Typen belegen, dass nicht nur auf Basis von Wasserstoffbrücken Erbinformation gespeichert und wieder abgerufen werden kann. Dieser Aufsatz beschreibt die jüngsten Entwicklungen auf dem Gebiet der UBPs sowie ihre mögliche Anwendungen, einschließlich der Erweiterung der Evolution um den Einbau neuer Funktionalität sowie der Erschaffung semisynthetischen Lebens, das zusätzliche Information speichern kann.
Co-reporter:Ramkrishna Adhikary ; Jörg Zimmermann ; Jian Liu ; Ryan P. Forrest ; Tesia D. Janicki ; Philip E. Dawson ; Steven A. Corcelli
Journal of the American Chemical Society 2014 Volume 136(Issue 39) pp:13474-13477
Publication Date(Web):September 16, 2014
DOI:10.1021/ja503107h
Many residues within proteins adopt conformations that appear to be stabilized by interactions between an amide N–H and the amide N of the previous residue. To explore whether these interactions constitute hydrogen bonds, we characterized the IR stretching frequencies of deuterated variants of proline and the corresponding carbamate, as well as the four proline residues of an Src homology 3 domain protein. The CδD2 stretching frequencies are shifted to lower energies due to hyperconjugation with Ni electron density, and engaging this density via protonation or the formation of the Ni+1–H···Ni interaction ablates this hyperconjugation and thus induces an otherwise difficult to explain blue shift in the C–D absorptions. Along with density functional theory calculations, the data reveal that the Ni+1–H···Ni interactions constitute H-bonds and suggest that they may play an important and previously underappreciated role in protein folding, structure, and function.
Co-reporter:Rodrigo A. Rodriguez ; Danielle Barrios Steed ; Yu Kawamata ; Shun Su ; Peter A. Smith ; Tyler C. Steed ; Floyd E. Romesberg ;Phil S. Baran
Journal of the American Chemical Society 2014 Volume 136(Issue 43) pp:15403-15413
Publication Date(Web):October 20, 2014
DOI:10.1021/ja508632y
Antibiotic-resistant bacteria present an ongoing challenge to both chemists and biologists as they seek novel compounds and modes of action to out-maneuver continually evolving resistance pathways, especially against Gram-negative strains. The dimeric pyrrole–imidazole alkaloids represent a unique marine natural product class with diverse primary biological activity and chemical architecture. This full account traces the strategy used to develop a second-generation route to key spirocycle 9, culminating in a practical synthesis of the axinellamines and enabling their discovery as broad-spectrum antibacterial agents, with promising activity against both Gram-positive and Gram-negative bacteria. While their detailed mode of antibacterial action remains unclear, the axinellamines appear to cause secondary membrane destabilization and impart an aberrant cellular morphology consistent with the inhibition of normal septum formation. This study serves as a rare example of a natural product initially reported to be devoid of biological activity surfacing as an active antibacterial agent with an intriguing mode of action.
Co-reporter:Dr. Ramkrishna Adhikary;Dr. Jörg Zimmermann; Philip E. Dawson ; Floyd E. Romesberg
ChemPhysChem 2014 Volume 15( Issue 5) pp:849-853
Publication Date(Web):
DOI:10.1002/cphc.201400017
Abstract
A variety of IR-active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon-deuterium bonds (CD), cyano groups (CN), and azides (N3); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N-terminal Src homology 3 domain of the murine adapter protein Crk-II (nSH3) with CD bonds site-selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two-state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N3 probes at five of the same positions. In agreement with previous studies, the position-dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface-exposed positions. Thus, unlike CD labels, which are both sensitive to their environment and non-perturbative, CN and N3 probes should be used with caution.
Co-reporter:Thomas Lavergne ; Mélissa Degardin ; Denis A. Malyshev ; Henry T. Quach ; Kirandeep Dhami ; Phillip Ordoukhanian
Journal of the American Chemical Society 2013 Volume 135(Issue 14) pp:5408-5419
Publication Date(Web):April 2, 2013
DOI:10.1021/ja312148q
As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former; however, previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of 18 novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable, while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.
Co-reporter:Karin Betz ; Denis A. Malyshev ; Thomas Lavergne ; Wolfram Welte ; Kay Diederichs ; Floyd E. Romesberg ;Andreas Marx
Journal of the American Chemical Society 2013 Volume 135(Issue 49) pp:18637-18643
Publication Date(Web):November 27, 2013
DOI:10.1021/ja409609j
The genetic alphabet is composed of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq DNA polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson–Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the prechemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple postchemistry complexes in which the already formed unnatural base pair is positioned at the postinsertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the postinsertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by deintercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.
Co-reporter:Lingjun Li ; Mélissa Degardin ; Thomas Lavergne ; Denis A. Malyshev ; Kirandeep Dhami ; Phillip Ordoukhanian
Journal of the American Chemical Society 2013 Volume 136(Issue 3) pp:826-829
Publication Date(Web):October 23, 2013
DOI:10.1021/ja408814g
We synthesized a panel of unnatural base pairs whose pairing depends on hydrophobic and packing forces and identify dTPT3-dNaM, which is PCR amplified with a natural base pair-like efficiency and fidelity. In addition, the dTPT3 scaffold is uniquely tolerant of attaching a propargyl amine linker, resulting in the dTPT3PA-dNaM pair, which is amplified only slightly less well. The identification of dTPT3 represents significant progress toward developing an unnatural base pair for the in vivo expansion of an organism’s genetic alphabet and for a variety of in vitro biotechnology applications where it is used to site-specifically label amplified DNA, and it also demonstrates for the first time that hydrophobic and packing forces are sufficient to mediate natural-like replication.
Co-reporter:Jian Liu, Peter A. Smith, Danielle Barrios Steed, Floyd Romesberg
Bioorganic & Medicinal Chemistry Letters 2013 Volume 23(Issue 20) pp:5654-5659
Publication Date(Web):15 October 2013
DOI:10.1016/j.bmcl.2013.08.026
New antibiotics are needed, and one source may be ‘latent’ antibiotics, natural products whose once broad-spectrum activity is currently limited by the evolution of resistance in nature. We have identified a potential class of latent antibiotics, the arylomycins, which are lipopeptides with a C-terminal macrocycle that target signal peptidase and whose spectrum is limited by a resistance-conferring mutation in many bacteria. Herein, we report the synthesis and evaluation of several arylomycin derivatives, and demonstrate that both C-terminal homologation with a glycyl aldehyde and addition of a positive charge to the macrocycle increase the activity and spectrum of the arylomycin scaffold.
Co-reporter:Dr. Zhengtao Li;Dr. Thomas Lavergne;Denis A. Malyshev;Dr. Jörg Zimmermann;Dr. Ramkrishna Adhikary;Kireep Dhami;Dr. Phillip Ordoukhanian;Dr. Zhelin Sun; Jie Xiang; Floyd E. Romesberg
Chemistry - A European Journal 2013 Volume 19( Issue 42) pp:14205-14209
Publication Date(Web):
DOI:10.1002/chem.201302496
Abstract
A class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM were developed. To explore the use of these pairs to produce site-specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA by Click chemistry is reported. With the d5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the dMMO2 and dDMO analogues, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICSCO–dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps “evolution” of nanomaterials.
Co-reporter:Ramkrishna Adhikary, Jörg Zimmermann, Jian Liu, Philip E. Dawson, and Floyd E. Romesberg
The Journal of Physical Chemistry B 2013 Volume 117(Issue 42) pp:13082-13089
Publication Date(Web):July 9, 2013
DOI:10.1021/jp402772x
Electrostatic and conformational heterogeneity make central contributions to protein function, but their experimental characterization requires a combination of spatial and temporal resolution that is challenging to achieve. Src homology 3 (SH3) domains mediate protein–protein interactions, and NMR studies have demonstrated that most possess conformational heterogeneity, which could be critical for their function. Here, we use the IR absorptions of carbon–deuterium (C–D) bonds site-selectively incorporated throughout the N-terminal SH3 domain from the murine adapter protein Crk-II to characterize its different microenvironments with high spatial and temporal resolution. The C–D absorptions are only differentiated in the folded state of the protein where they show evidence of significant environmental heterogeneity. However, the spectra of the folded state are independent of temperature, and upon thermal denaturation the protein undergoes a single, global unfolding transition. While some evidence of conformational heterogeneity is found within the peptide backbone, the majority of the environmental heterogeneity appears to result from electrostatics.
Co-reporter:Yun Xuan Tan and Floyd E. Romesberg
MedChemComm 2012 vol. 3(Issue 8) pp:916-925
Publication Date(Web):13 Apr 2012
DOI:10.1039/C2MD20043K
The increasing trend of antibiotic resistance in bacterial pathogens has driven the need for new classes of antibiotics acting by novel mechanisms. The arylomycins are natural product antibiotics that inhibit bacterial type I signal peptidase (SPase), an endoprotease that is required for the translocation of most proteins across the cytoplasmic membrane. SPase is a promising antibiotic target due to its essentiality and its conserved and accessible active site. However, the initial reported spectrum of arylomycin activity was surprisingly narrow. The total synthesis of several members of this fascinating family of natural products has allowed for a more thorough study of its activity. It has been shown that their spectrum of activity is much broader than previously believed, and that their activity is generally limited not by factors intrinsic to their scaffold or to SPase as a target, but rather, by specific mutations in SPase. An interesting possibility is that past competition between producer and susceptible strains of bacteria may have led to the development of much of the intrinsic resistance observed today. The arylomycins may thus represent “latent” antibiotics, natural product antibiotics whose scaffolds once possessed potent and broad-spectrum activity, and are more likely to be optimized to again have potent and broad spectrum activity, than candidate scaffolds that have never been antibiotics.
Co-reporter:Denis A. Malyshev;Kirandeep Dhami;Thomas Lavergne;Henry T. Quach;Ali Torkamani;Phillip Ordoukhanian
PNAS 2012 Volume 109 (Issue 30 ) pp:12005-12010
Publication Date(Web):2012-07-24
DOI:10.1073/pnas.1205176109
The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all
life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic
biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and
dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence
contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity.
To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing
the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with
high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is
functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter
genetic alphabet.
Co-reporter:Dr. Thomas Lavergne;Denis A. Malyshev ;Dr. Floyd E. Romesberg
Chemistry - A European Journal 2012 Volume 18( Issue 4) pp:1231-1239
Publication Date(Web):
DOI:10.1002/chem.201102066
Abstract
Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d5SICS and analogues of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new dMMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, dNaM–d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure–activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogues with the exception of dNaM. We also found that dNaM–d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.
Co-reporter:Wayne Yu, Phillip E. Dawson, Jörg Zimmermann, and Floyd E. Romesberg
The Journal of Physical Chemistry B 2012 Volume 116(Issue 22) pp:6397-6403
Publication Date(Web):May 24, 2012
DOI:10.1021/jp303521t
We report a residue-specific characterization of the thermal unfolding mechanism of ferric horse heart cytochrome c using C–D bonds site-specifically incorporated at residues dispersed throughout three different structural elements within the protein. As the temperature increases, Met80 first dissociates from the heme center, and the protein populates a folding intermediate before transitioning to a solvent exposed state. With further increases in temperature, the C-terminal helix frays and then loses structure along with the core of the protein. Interestingly, the data also reveal that the state populated at high temperature retains some structure and possibly represents a molten globule. Elucidation of the detailed unfolding mechanism and the structure of the associated molten globule, both of which represent challenges to conventional techniques, highlights the utility of the C–D technique.
Co-reporter:Jian Liu ; Chuanyun Luo ; Peter A. Smith ; Jodie K. Chin ; Malcolm G. P. Page ; Mark Paetzel
Journal of the American Chemical Society 2011 Volume 133(Issue 44) pp:17869-17877
Publication Date(Web):October 14, 2011
DOI:10.1021/ja207318n
Glycosylation of natural products, including antibiotics, often plays an important role in determining their physical properties and their biological activity, and thus their potential as drug candidates. The arylomycin class of antibiotics inhibits bacterial type I signal peptidase and is comprised of three related series of natural products with a lipopeptide tail attached to a core macrocycle. Previously, we reported the total synthesis of several A series derivatives, which have unmodified core macrocycles, as well as B series derivatives, which have a nitrated macrocycle. We now report the synthesis and biological evaluation of lipoglycopeptide arylomycin variants whose macrocycles are glycosylated with a deoxy-α-mannose substituent, and also in some cases hydroxylated. The synthesis of the derivatives bearing each possible deoxy-α-mannose enantiomer allowed us to assign the absolute stereochemistry of the sugar in the natural product and also to show that while glycosylation does not alter antibacterial activity, it does appear to improve solubility. Crystallographic structural studies of a lipoglycopeptide arylomycin bound to its signal peptidase target reveal the molecular interactions that underlie inhibition and also that the mannose is directed away from the binding site into solvent which suggests that other modifications may be made at the same position to further increase solubility and thus reduce protein binding and possibly optimize the pharmacokinetics of the scaffold.
Co-reporter:Young Jun Seo ; Denis A. Malyshev ; Thomas Lavergne ; Phillip Ordoukhanian
Journal of the American Chemical Society 2011 Volume 133(Issue 49) pp:19878-19888
Publication Date(Web):October 8, 2011
DOI:10.1021/ja207907d
Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the postamplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications.
Co-reporter:Tucker C. Roberts ; Mark A. Schallenberger ; Jian Liu ; Peter A. Smith
Journal of Medicinal Chemistry 2011 Volume 54(Issue 14) pp:4954-4963
Publication Date(Web):June 1, 2011
DOI:10.1021/jm1016126
While most clinically used antibiotics were derived from natural products, the isolation of new broad-spectrum natural products has become increasingly rare and narrow-spectrum agents are typically deemed unsuitable for development because of intrinsic limitations of their scaffold or target. However, it is possible that the spectrum of a natural product antibiotic might be limited by specific resistance mechanisms in some bacteria, such as target mutations, and the spectra of such “latent” antibiotics might be reoptimized by derivatization, just as has been done with clinically deployed antibiotics. We recently showed that the spectrum of the arylomycin natural product antibiotics, which act via the novel mechanism of inhibiting type I signal peptidase, is broader than previously believed and that resistance in several key human pathogens is due to the presence of a specific Pro residue in the target peptidase that disrupts interactions with the lipopeptide tail of the antibiotic. To begin to test whether this natural resistance might be overcome by derivatization, we synthesized analogues with altered lipopeptide tails and identified several with an increased spectrum of activity against S. aureus. The data support the hypothesis that the arylomycins are latent antibiotics, suggest that their spectrum may be optimized by derivatization, and identify a promising scaffold upon which future optimization efforts might focus.
Co-reporter:Tucker C. Roberts, Peter A. Smith, and Floyd E. Romesberg
Journal of Natural Products 2011 Volume 74(Issue 5) pp:956-961
Publication Date(Web):May 5, 2011
DOI:10.1021/np200163g
Antibiotics are virtually always isolated as families of related compounds, but the evolutionary forces underlying the observed diversity are generally poorly understood, and it is not even clear whether they are all expected to be biologically active. The arylomycin class of antibiotics is comprised of three related families that are differentiated by nitration, glycosylation, and hydroxylation of a conserved core scaffold. Previously, we reported the total synthesis of an A series member, arylomycin A2, as well as the A series derivative arylomycin C16 and showed that both are active against a broader spectrum of bacteria than previously appreciated. We now report the total synthesis of a B series analogue, arylomycin B-C16, and its aromatic amine derivative. While the aromatic amine loses activity against all bacteria tested, the B series compound shows activities that are similar to the A series compounds, except that it also gains activity against the important pathogen Streptococcus agalactiae.
Co-reporter:Dr. Jörg Zimmermann;Megan C. Thielges;Dr. Young Jun Seo; Philip E. Dawson ; Floyd E. Romesberg
Angewandte Chemie International Edition 2011 Volume 50( Issue 36) pp:8333-8337
Publication Date(Web):
DOI:10.1002/anie.201101016
Co-reporter:Dr. Jörg Zimmermann;Megan C. Thielges;Dr. Young Jun Seo; Philip E. Dawson ; Floyd E. Romesberg
Angewandte Chemie 2011 Volume 123( Issue 36) pp:8483-8487
Publication Date(Web):
DOI:10.1002/ange.201101016
Co-reporter:Jörg Zimmermann, Megan C. Thielges, Wayne Yu, Philip E. Dawson, and Floyd E. Romesberg
The Journal of Physical Chemistry Letters 2011 Volume 2(Issue 5) pp:412-416
Publication Date(Web):February 8, 2011
DOI:10.1021/jz200012h
Carbon−deuterium (C−D) bonds are nonperturbative spectroscopic probes that absorb in a region of the IR spectrum that is free of other protein absorptions. We explore the use of these probes under time-resolved conditions to follow the unfolding of cytochrome c from a photostationary state that accumulates after CO is photodissociated from the protein’s heme prosthetic group. Our results clearly show that C−D bonds are well-suited to characterize protein folding and dynamics.Keywords: cytochrome c; heme ligation; IR spectroscopy; isotopic substitution; unfolding;
Co-reporter:Peter A. Smith, Tucker C. Roberts, Floyd E. Romesberg
Chemistry & Biology 2010 Volume 17(Issue 11) pp:1223-1231
Publication Date(Web):24 November 2010
DOI:10.1016/j.chembiol.2010.09.009
Novel classes of broad-spectrum antibiotics are needed to treat multidrug-resistant pathogens. The arylomycin class of natural products inhibits a promising antimicrobial target, type I signal peptidase (SPase), but upon initial characterization appeared to lack whole-cell activity against most pathogens. Here, we show that Staphylococcus epidermidis, which is sensitive to the arylomycins, evolves resistance via mutations in SPase and that analogous mutations are responsible for the natural resistance of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We identify diverse bacteria lacking these mutations and demonstrate that most are sensitive to the arylomycins. The results illustrate that the arylomycins have a broad-spectrum of activity and are viable candidates for development into therapeutics. The results also raise the possibility that naturally occurring resistance may have masked other natural product scaffolds that might be developed into therapeutics.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (309 K)Download as PowerPoint slideHighlights► The arylomycins have broad-spectrum antibiotic activity ► Resistance in key pathogens is mediated by naturally occurring target mutations ► The arylomycin scaffold is promising for development as a broad-spectrum antibiotic ► Natural resistance may prevent the identification of novel classes of antibiotics
Co-reporter:AaronM. Leconte;MahaP. Patel;LaurynE. Sass Dr.;Peter McInerney Dr.;Mirna Jarosz Dr.;Li Kung Dr.;JaysonL. Bowers Dr.;PhilipR. Buzby Dr.;J.William Efcavitch Dr.;FloydE. Romesberg Dr.
Angewandte Chemie 2010 Volume 122( Issue 34) pp:6057-6060
Publication Date(Web):
DOI:10.1002/ange.201001607
Co-reporter:AaronM. Leconte;MahaP. Patel;LaurynE. Sass Dr.;Peter McInerney Dr.;Mirna Jarosz Dr.;Li Kung Dr.;JaysonL. Bowers Dr.;PhilipR. Buzby Dr.;J.William Efcavitch Dr.;FloydE. Romesberg Dr.
Angewandte Chemie International Edition 2010 Volume 49( Issue 34) pp:5921-5924
Publication Date(Web):
DOI:10.1002/anie.201001607
Co-reporter:Denis A. Malyshev;Danielle A. Pfaff;Shannon I. Ippoliti;Dr. Gil Tae Hwang; Tammy J. Dwyer; Floyd E. Romesberg
Chemistry - A European Journal 2010 Volume 16( Issue 42) pp:12650-12659
Publication Date(Web):
DOI:10.1002/chem.201000959
Abstract
As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge-on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue dMMO2, which forms an unnatural base pair with the nucleotide analogue d5SICS. Replacing the para-methyl substituent of dMMO2 with an annulated furan ring (yielding dFMO) has a dramatically negative effect on replication, while replacing it with a methoxy (dDMO) or with a thiomethyl group (dTMO) improves replication in both steady-state assays and during PCR amplification. Thus, dTMO–d5SICS, and especially dDMO–d5SICS, represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure–activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental dMMO2–d5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.
Co-reporter:Matthew E. Cremeens ; Jörg Zimmermann ; Wayne Yu ; Philip E. Dawson
Journal of the American Chemical Society 2009 Volume 131(Issue 16) pp:5726-5727
Publication Date(Web):April 7, 2009
DOI:10.1021/ja900505e
Structural heterogeneity is thought to be inherent in many proteins and may be important for their folding and/or function. However, it is difficult to detect by conventional methods. Carbon−deuterium bonds are environmentally sensitive, nonperturbative probes of protein environments whose observation and characterization are facilitated by their unique stretching absorption frequency in an otherwise unobscured region of the IR spectrum. We demonstrate that deuterium atoms incorporated at Cα backbone positions (Cα−D bonds) are sensitive to the local backbone structure and thus may be used not only to detect structural heterogeneity but also to help characterize it structurally. Density functional theory calculations are used to predict that Cα−D bonds of glycine are sensitive to their local structure, with the absorptions red-shifted for an extended β-sheet relative to γ- and α-helix-like turns. These predictions are confirmed using the N-terminal Src homology 3 (nSH3) domain from the human CrkII adaptor protein, whose function as a signaling domain may require structural heterogeneity. Four nSH3 variants were synthesized in which individual glycine residues were site-specifically modified with CαD2 glycine residues. Not only were the Cα−D bonds incorporated within the β-sheet of nSH3 more red-shifted than those incorporated within loops, but the data also reveal that nSH3 populates at least two discrete β-sheet core structures. Moreover, the data suggest that the folded core of nSH3 may be less ordered than previously believed and also that the unfolded state may be more ordered than previously thought, and both of these factors may influence the folding and function of these important signaling domains. The C−D-based IR technique should be generally useful in the characterization of structure and heterogeneity of both folded and unfolded states.
Co-reporter:Denis A. Malyshev ; Young Jun Seo ; Phillip Ordoukhanian
Journal of the American Chemical Society 2009 Volume 131(Issue 41) pp:14620-14621
Publication Date(Web):September 29, 2009
DOI:10.1021/ja906186f
Expansion of the genetic alphabet with a third base pair would lay the foundation for a semisynthetic organism with an expanded genetic code and also have immediate in vitro applications. Previously, the unnatural base pairs formed between d5SICS and either dNaM or dMMO2 were shown to be well-replicated by DNA polymerases under steady-state conditions and also transcribed by T7 RNA polymerase efficiently in either direction. We now demonstrate that DNA containing either the d5SICS−dNaM or d5SICS−dMMO2 unnatural base pair may be amplified by PCR with fidelities and efficiencies that approach those of fully natural DNA. These results further demonstrate that the determinants of a functional unnatural base pair may be designed into predominantly hydrophobic nucleobases with no structural similarity to the natural purines or pyrimidines. Importantly, the results reveal that the unnatural base pairs may function within an expanded genetic alphabet and make possible many in vitro applications.
Co-reporter:Megan C. Thielges ; Jörg Zimmermann
Journal of the American Chemical Society 2009 Volume 131(Issue 17) pp:6054-6055
Publication Date(Web):April 10, 2009
DOI:10.1021/ja810155s
Horse heart cytochrome c (cyt c) has emerged as a paradigm for the study of protein folding, in large part because the covalently bound heme facilitates its characterization. The folding of reduced cyt c induced by photodissociation of CO from the CO-bound unfolded protein has been extensively studied. Following a nanosecond light pulse, four transitions with time constants of approximately 1−5, 50−100, 200−500, and 1000−10000 μs have been resolved. While originally thought to be associated with CO rebinding to two different partially folded states of cyt c, the two slower processes are now understood to reflect the bimolecular reassociation of CO followed by religation of His18, which by the base elimination mechanism is induced to dissociate after CO photolysis. Thus, it turns out that the two longer time constants do not report on protein folding but instead reflect the complexity of heme ligation. The two shorter time constants have been attributed to ligation at the heme center by Met65 or Met80 and His33 or His26 and have been used to estimate interchain diffusion rates of the protein. Here, to unambiguously determine the post-photodissociation steps involving CO, we have monitored the CO vibration following photodissociation with step-scan FT-IR spectroscopy. We have found that like the longer time scale processes, the 50−100 μs time scale process is associated not with protein dynamics but with CO ligand dynamics. The data clearly demonstrate that whatever the origins of the spectral changes, they clearly involve CO rebinding or changes in the environment of an already bound CO ligand. We speculate that the observed changes reflect His18 religation after fast geminate recombination of the CO. The data suggest that the associated time constant should not be used as a measure of interchain diffusion, and the results emphasize the importance of studying protein folding with probes that have inherently high structural resolution.
Co-reporter:David A. Harris, Michael E. Powers, Floyd E. Romesberg
Bioorganic & Medicinal Chemistry Letters 2009 Volume 19(Issue 14) pp:3787-3790
Publication Date(Web):15 July 2009
DOI:10.1016/j.bmcl.2009.04.034
We report the first synthesis of a 5S penem, known to bind bacterial type I signal peptidase, from the commercially available and inexpensive 6-aminopenicillanic acid. We report the first in vivo activity of the compound and use structure–activity relationship studies to begin to define the determinants of signal peptidase binding and also to begin to optimize the penem as an antibiotic.The synthesis of a 5S penem inhibitor of bacterial signal peptidase is reported and shown to have activity against human pathogens and to be potentially optimizable for spectrum and activity.
Co-reporter:Dan Groff;MeganC. Thielges;Susan Cellitti;PeterG. Schultz Dr. ;FloydE. Romesberg Dr.
Angewandte Chemie International Edition 2009 Volume 48( Issue 19) pp:3478-3481
Publication Date(Web):
DOI:10.1002/anie.200806239
Co-reporter:Jörg Zimmermann, Kenan Gundogdu, Matthew E. Cremeens, Jigar N. Bandaria, Gil Tae Hwang, Megan C. Thielges, Christopher M. Cheatum and Floyd E. Romesberg
The Journal of Physical Chemistry B 2009 Volume 113(Issue 23) pp:7991-7994
Publication Date(Web):May 14, 2009
DOI:10.1021/jp900516c
The spectral position of C−D stretching absorptions in the so-called “transparent window” of protein absorption (1800−2300 cm−1) makes them well suited as probes of protein dynamics with high temporal and structural resolution. We have previously incorporated single deuterated amino acids into proteins to site-selectively follow protein folding and ligand binding by steady-state FT IR spectroscopy. Ultimately, our goal is to use C−D bonds as probes in time-resolved IR spectroscopy to study dynamics and intramolecular vibrational energy redistribution (IVR) in proteins. As a step toward this goal, we now present the first time-resolved experiments characterizing the population and dephasing dynamics of selectively excited C−D bonds in a deuterated amino acid. Three differently deuterated, Boc-protected leucines were selected to systematically alter the number of additional C−D bonds that may mediate IVR out of the initially populated bright C−D stretching mode. Three-pulse photon echo experiments show that the steady-state C−D absorption linewidths are broadened by both homogeneous and inhomogeneous effects, and transient grating experiments reveal that IVR occurs on a subpicosecond time scale and is nonstatistical. The results have important implications for the interpretation of steady-state C−D spectra and demonstrate the potential utility of C−D bonds as probes of dynamics and IVR within a protein.
Co-reporter:Young Jun Seo Dr. Dr.
ChemBioChem 2009 Volume 10( Issue 14) pp:
Publication Date(Web):
DOI:10.1002/cbic.200990060
Co-reporter:Young Jun Seo Dr. Dr.
ChemBioChem 2009 Volume 10( Issue 14) pp:2394-2400
Publication Date(Web):
DOI:10.1002/cbic.200900413
Abstract
An unnatural base pair that is replicated and transcribed with good efficiency would lay the foundation for the long term goal of creating a semisynthetic organism, but also would have immediate in vitro applications, such as the enzymatic synthesis of site-specifically modified DNA and/or RNA. One of the most promising of the unnatural base pairs that we have identified is formed between d5SICS and dMMO2. The ortho substituents of these nucleotides are included to facilitate unnatural base pair extension, presumably by forming a hydrogen-bond with the polymerase, but the synthesis of the unnatural base pair still requires optimization. Recently, we have shown that meta and/or para substituents within the dMMO2 scaffold can facilitate unnatural base pair synthesis, although the mechanism remains unclear. To explore this issue, we synthesized and evaluated several dMMO2 derivatives with meta-chlorine, -bromine, -iodine, -methyl, or -propinyl substituents. Complete characterization of unnatural base pair and mispair synthesis and extension reveal that the modifications have large effects only on the efficiency of unnatural base pair synthesis and that the effects likely result from a combination of changes in steric interactions, polarity, and polarizability. The results also suggest that functionalized versions of the propinyl moiety of d5PrM should serve as suitable linkers to site-specifically incorporate other chemical functionalities into DNA. Similar modifications of d5SICS should allow labeling of DNA with two different functionalities, and the previously demonstrated efficient transcription of the unnatural base pair suggests that derivatives might similarly enable site-specific labeling of RNA.
Co-reporter:Megan C. Thielges, Jörg Zimmermann, Wayne Yu, Masayuki Oda and Floyd E. Romesberg
Biochemistry 2008 Volume 47(Issue 27) pp:
Publication Date(Web):June 13, 2008
DOI:10.1021/bi800374q
The production of antibodies that selectively bind virtually any foreign compound is the hallmark of the immune system. While much is understood about how sequence diversity contributes to this remarkable feat of molecular recognition, little is known about how sequence diversity impacts antibody dynamics, which is also expected to contribute to molecular recognition. Toward this goal, we examined a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we selected six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. Sequencing revealed that two pairs of antibodies employ homologous heavy chains that were derived from common germline genes, while the other two heavy chains and all six of the light chains were derived from different germline genes and are not homologous. Interestingly, more than half of all the somatic mutations acquired during affinity maturation among the six antibodies are located in positions unlikely to contact fluorescein directly. To quantify and compare the dynamics of the antibody−fluorescein complexes, three-pulse photon echo peak shift and transient grating spectroscopy were employed. All of the antibodies exhibited motions on three distinct time scales, ultrafast motions on the <100 fs time scale, diffusive motions on the picosecond time scale, and motions that occur on time scales longer than nanoseconds and thus appear static. However, the exact frequency of the picosecond time scale motion and the relative contribution of the different motions vary significantly among the antibody−chromophore complexes, revealing a high level of dynamic diversity. Using a hierarchical model, we relate the data to features of the antibodies’ energy landscapes as well as their flexibility in terms of elasticity and plasticity. In all, the data provide a consistent picture of antibody flexibility, which interestingly appears to be correlated with binding entropy as well as with germline gene use and the mutations introduced during affinity maturation. The data also provide a gauge of the dynamic diversity of the antibody repertoire and suggest that this diversity might contribute to molecular recognition by facilitating the recognition of the broadest range of foreign molecules.
Co-reporter:Patrick Weinkam, Jörg Zimmermann, Laura B. Sagle, Shigeo Matsuda, Philip E. Dawson, Peter G. Wolynes and Floyd E. Romesberg
Biochemistry 2008 Volume 47(Issue 51) pp:13470-13480
Publication Date(Web):November 26, 2008
DOI:10.1021/bi801223n
The alkaline-induced structural transitions of ferricytochrome c have been studied intensively as a model for how changes in metal ligation contribute to protein function and folding. Previous studies have demonstrated that multiple non-native species accumulate with increasing pH. Here, we used a combination of experiments and simulations to provide a high-resolution view of the changes associated with increasing alkaline conditions. Alkaline-induced transitions were characterized under equilibrium conditions by following changes in the IR absorptions of carbon−deuterium chromophores incorporated at Leu68, Lys72, Lys73, Lys79, and Met80. The data suggest that at least four intermediates are formed as the pH is increased prior to complete unfolding of the protein. The first alkaline transition observed appears to be driven by a single deprotonation and occurs with a midpoint of pH 8.8, but surprisingly, the intermediate formed does not appear to be one of the well-characterized lysine misligates. At higher pH, second and third deprotonations, with a combined apparent midpoint pH of 10.2, induce transitions to Lys73- or Lys79-misligated species. Interestingly, the lysine misligates appear to undergo iron reduction by the coordinated amine. A transition from the lysine misligates to another intermediate, likely a hydroxide-misligated species, is associated with a fourth deprotonation and a midpoint of pH 10.7. Finally, the protein loses tertiary structure with a fifth deprotonation that occurs with a midpoint of pH 12.7. Native topology-based models with enforced misligation are employed to help understand the structures of the observed intermediates.
Co-reporter:Yoshiyuki Hari Dr.;Gil Tae Hwang Dr.;Aaron M. Leconte;Nicolas Joubert;Michal Hocek Dr. Dr.
ChemBioChem 2008 Volume 9( Issue 17) pp:2796-2799
Publication Date(Web):
DOI:10.1002/cbic.200800577
Co-reporter:Bryan M. O'Neill;Shawn J. Szyjka;Ewa T. Lis;Aaron O. Bailey;John R. Yates III;Oscar M. Aparicio
PNAS 2007 104 (22 ) pp:9290-9295
Publication Date(Web):2007-05-29
DOI:10.1073/pnas.0703252104
Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication
forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53
is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation
by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is
not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms
a complex with Psy2 (Pph3–Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from,
methylmethane sulfonate-mediated DNA damage. In the absence of Pph3–Psy2, Rad53 dephosphorylation and the resumption of DNA
synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication
forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the
presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation
of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.
Co-reporter:Gil Tae Hwang Dr.;Aaron M. Leconte
ChemBioChem 2007 Volume 8(Issue 13) pp:
Publication Date(Web):23 JUL 2007
DOI:10.1002/cbic.200700308
Recently much effort has been focused on designing unnatural base pairs that are stable and replicated by DNA polymerases with high efficiency and fidelity. This work has helped to identify a variety of nucleobase properties that are capable of mediating the required interbase interactions in the absence of Watson–Crick hydrogen-bonding complementarity. These properties include shape complementarity, the presence of a suitably positioned hydrogen-bond donor in the developing minor groove, and fluorine substitution. In order to help characterize how each factor contributes to base pairing stability and replication, we synthesized and characterized three fluoro-substituted pyridone nucleoside analogues, 3 FP, 4 FP, and 5 FP. Generally, we found that the specific fluorine substitution pattern of the analogues had little impact on unnatural pair or mispair stability, with the exception of mispairs with dG, which were also the most stable. The mispair between dG and 3 FP was less stable than that with 4 FP or 5 FP, which likely resulted from specific interbase interactions. While fluorine substitution had little impact on the synthesis of the unnatural base pairs, it significantly enhanced mispairing with dG. Remarkably, the mispair between dG and 3 FP was the most efficiently synthesized, due to a favorable entropy of activation, which possibly resulted from the displacement of water molecules from dG in the phosphoryl transfer transition state. The more efficient synthesis of the 3 FP–dG mispair, despite its being the least stable of the three, suggests that the determinants of synthesis and stability are distinct. Finally, we found that fluorine substitution significantly increased the rate at which the pyridone-based unnatural base pairs were extended; this suggests that both minor groove hydrogen-bond acceptors and fluorine substituents could be used to simultaneously optimize unnatural base pairs.
Co-reporter:Yoonkyung Kim;Aaron M. Leconte;Yoshiyuki Hari Dr.
Angewandte Chemie 2006 Volume 118(Issue 46) pp:
Publication Date(Web):31 OCT 2006
DOI:10.1002/ange.200602579
Für den Aufbau nichtnatürlicher Basenpaare wurden Pyridyl-Nucleosidanaloga synthetisiert und charakterisiert (siehe Strukturen). Ein α-glycosidisches N-Atom wirkt als H-Brückenacceptor, der die Paarung mit natürlichen Nucleobasen nicht signifikant erleichtert. Es scheint jedoch in der kleinen Furche H-Brücken mit H2O-Molekülen und DNA-Polymerasen zu bilden, die die Stabilität bzw. Replikation des nichtnatürlichen Basenpaars optimieren.
Co-reporter:Aaron M. Leconte;Shigeo Matsuda;Gil Tae Hwang Dr.
Angewandte Chemie 2006 Volume 118(Issue 26) pp:
Publication Date(Web):30 MAY 2006
DOI:10.1002/ange.200601272
Ein übliches Problem bei hydrophoben nichtnatürlichen Basenpaaren – die ineffiziente Verlängerung an so einem Basenpaar – wird durch die Verwendung von Methylpyridongruppen vermindert, was zugleich ein anderes häufiges Problem nichtnatürlicher Basen eingrenzt: Fehlpaarungen mit Desoxyadenosin.
Co-reporter:Jörg Zimmermann;Erin L. Oakman;Ian F. Thorpe;Xinghua Shi;Paul Abbyad;Charles L. Brooks III;Steven G. Boxer
PNAS 2006 Volume 103 (Issue 37 ) pp:13722-13727
Publication Date(Web):2006-09-12
DOI:10.1073/pnas.0603282103
The evolution of proteins with novel function is thought to start from precursor proteins that are conformationally heterogeneous.
The corresponding genes may be duplicated and then mutated to select and optimize a specific conformation. However, testing
this idea has been difficult because of the challenge of quantifying protein flexibility and conformational heterogeneity
as a function of evolution. Here, we report the characterization of protein heterogeneity and dynamics as a function of evolution
for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics
simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations
to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein.
This study demonstrates how protein dynamics may be tailored by evolution and has important implications for our understanding
of how novel protein functions are evolved.
Co-reporter:Aaron M. Leconte
and
Floyd E. Romesberg
Nature 2006 444(7119) pp:553
Publication Date(Web):
DOI:10.1038/444553a
Slipping in extra benzene rings creates a broader DNA double helix that is similar to, but different from, natural DNA. Importantly, it can encode more genetic information — and that could have wide implications.
Co-reporter:Yoonkyung Kim;Aaron M. Leconte;Yoshiyuki Hari Dr.
Angewandte Chemie International Edition 2006 Volume 45(Issue 46) pp:
Publication Date(Web):31 OCT 2006
DOI:10.1002/anie.200602579
In the groove: In an effort to develop unnatural base pairs, pyridyl nucleoside analogues were synthesized and characterized (see structures). An α-glycosidic nitrogen atom provides an H-bond acceptor that does not significantly facilitate pairing with natural nucleobases. However, it forms minor-groove H-bonds with water molecules and DNA polymerases that optimize the stability and replication, respectively, of the unnatural base pair.
Co-reporter:Aaron M. Leconte;Shigeo Matsuda;Gil Tae Hwang Dr.
Angewandte Chemie International Edition 2006 Volume 45(Issue 26) pp:
Publication Date(Web):30 MAY 2006
DOI:10.1002/anie.200601272
Getting on with it: A common problem with hydrophobic non-natural base pairs—the inefficient extension following such a base pair—is reduced by using the methylpyridone scaffold, which simultaneously limits another frequent problem of non-natural bases: mispairs with deoxyadenosine.
Co-reporter:Jun Yin;Ralph Jimenez;Georgina Salazar;Taiha Joo
PNAS 2004 Volume 101 (Issue 11 ) pp:3803-3808
Publication Date(Web):2004-03-16
DOI:10.1073/pnas.0305745101
While it is accepted that protein flexibility plays a role in protein folding, catalysis, and molecular recognition, few techniques
are capable of the rigorous measurement of protein motions required to quantify flexibility. Three-pulse photon echo shift
spectroscopy can be used to measure the time scale of protein motions, and we have used this technique, along with steady-state
spectroscopy and binding and structural data, to examine the immunological evolution of protein flexibility in an anti-fluorescein
antibody. Two light chain somatic mutations increase affinity for fluorescein by 12-fold but also significantly affect flexibility.
Specifically, a rigidification of the protein is seen in each of three observable motions; two slower motions undergo decreased
amplitudes of displacement, by 3- and 20-fold, respectively, in response to an applied force, and the distribution associated
with the amplitude of a faster motion is narrowed upon somatic mutation. The somatic mutations appear to rigidify the antibody-fluorescein
complex by more strongly anchoring fluorescein to the protein and by more tightly packing the complex. The data demonstrate
that in addition to affinity, antibody dynamics are systematically manipulated during affinity maturation, and they imply
that the evolution of protein flexibility may be a central component of the immune response. The results also reflect the
type of protein rigidification that may be important for other biological interactions, such as protein-protein, protein-ligand
or protein-drug, and enzyme-substrate recognition.
Co-reporter:Allison A. Henry;Ralph Jimenez Dr.;Denise Hanway
ChemBioChem 2004 Volume 5(Issue 8) pp:
Publication Date(Web):2 AUG 2004
DOI:10.1002/cbic.200400013
E. coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm. Using the crystal structure as a guide, we mutated each protein residue that contacts the cofactor in an effort to identify the interactions responsible for this selective stabilization of the cofactor's excited state. Hydrogen bonding, packing, and electrostatic interactions were examined. Remarkably, a single residue, Glu109, appears to play an important, if not exclusive, role in inducing the observed red-shift. Thus, this protein, the simplest light-harvesting system known, appears to have evolved a remarkably simple mechanism to tune the photophysical properties of the antenna cofactor appropriately for biological function.
Co-reporter:Floyd E. Romesberg
ChemBioChem 2003 Volume 4(Issue 7) pp:
Publication Date(Web):27 JUN 2003
DOI:10.1002/cbic.200300572
Proteins on the move: The techniques of organic synthesis and molecular biology have been used to develop biological systems amenable to dynamic characterization by spectroscopic techniques (see picture). This review focuses on three novel systems designed to study tautomerization dynamics in DNA, the role of protein dynamics in function and folding, and molecular recognition within the immune system. These studies illustrate our efforts to understand biomolecule dynamics and to test whether nature has evolved biomolecules with specific dynamic properties tailored to perform specific biological functions.
Co-reporter:Ralph Jimenez;Georgina Salazar;Kim K. Baldridge;
Proceedings of the National Academy of Sciences 2003 100(1) pp:92-97
Publication Date(Web):December 23, 2002
DOI:10.1073/pnas.262411399
Photon echo spectroscopy has been used to measure the response of three antibody-binding sites to perturbation from electronic
excitation of a bound antigen, fluorescein. The three antibodies show motions that range in time scale from tens of femtoseconds
to nanoseconds. Relative to the others, one antibody, 4-4-20, possesses a rigid binding site that likely results from a short
and inflexible heavy chain complementarity-determining region 3 (HCDR3) loop and a critical Tyr that acts as a “molecular
splint,” rigidifying the antigen across its most flexible internal degree of freedom. The remaining two antibodies, 34F10
and 40G4, despite being generated against the same antigen, possess binding sites that are considerably more flexible. The
more flexible combining sites likely result from longer HCDR3 loops and a deletion in the light chain complementarity-determining
region 1 (LCDR1) that removes the critical Tyr residue. The binding site flexibilities may result in varying mechanisms of
antigen recognition including lock-and-key, induced-fit, and conformational selection.
Co-reporter:Chengzhi Yu Dr.;Allison A. Henry ;Peter G. Schultz
Angewandte Chemie International Edition 2002 Volume 41(Issue 20) pp:
Publication Date(Web):18 OCT 2002
DOI:10.1002/1521-3773(20021018)41:20<3841::AID-ANIE3841>3.0.CO;2-Q
More efficient and selective synthesis as well as more efficient extension by a DNA polymerase is achieved for unnatural base pairs upon heteroatom substitution. This has been demonstrated with modification of ICS (structure shown): Replacement of C6 with nitrogen and thio substitution at C10 provides the base SNICS, which forms stable self-pairs and can therefore be used for efficient unnatural base pair replication.
Co-reporter:Chengzhi Yu Dr.;Allison A. Henry ;Peter G. Schultz
Angewandte Chemie International Edition 2002 Volume 41(Issue 24) pp:
Publication Date(Web):12 DEC 2002
DOI:10.1002/anie.200290004
Co-reporter:Gang Xia;Liangjing Chen;Takashi Sera;Ming Fa;Peter G. Schultz;
Proceedings of the National Academy of Sciences 2002 99(10) pp:6597-6602
Publication Date(Web):2002-05-14
DOI:10.1073/pnas.102577799
The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space
of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity.
The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer
duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend
the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to
streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants
were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates
dNTP substrates.
Co-reporter:Chengzhi Yu Dr.;Allison A. Henry ;Peter G. Schultz
Angewandte Chemie 2002 Volume 114(Issue 24) pp:
Publication Date(Web):12 DEC 2002
DOI:10.1002/ange.200290056
Co-reporter:Markus Berger Dr.;Anthony K. Ogawa Dr.;Dustin L. McMinn Dr.;Yiqin Wu Dr.;Peter G. Schultz
Angewandte Chemie 2000 Volume 112(Issue 16) pp:
Publication Date(Web):11 AUG 2000
DOI:10.1002/1521-3757(20000818)112:16<3069::AID-ANGE3069>3.0.CO;2-K
Co-reporter:Megan C. Thielges, Jörg Zimmermann, Philip E. Dawson, Floyd E. Romesberg
Journal of Molecular Biology (24 April 2009) Volume 388(Issue 1) pp:159-167
Publication Date(Web):24 April 2009
DOI:10.1016/j.jmb.2009.02.059
Cytochrome c has served as a paradigm for the study of protein stability, folding, and molecular evolution, but it remains unclear how these aspects of the protein are related. For example, while the bovine and equine cytochromes c are known to have different stabilities, and possibly different folding mechanisms, it is not known how these differences arise from just three amino acid substitutions introduced during divergence. Using site-selectively incorporated carbon–deuterium bonds, we show that like the equine protein, bovine cytochrome c is induced to unfold by guanidine hydrochloride via a stepwise mechanism, but it does not populate an intermediate as is observed with the equine protein. The increased stability also results in more similar free energies of unfolding observed at different sites within the protein, giving the appearance of a more concerted mechanism. Furthermore, we show that the differences in stability and folding appear to result from a single amino acid substitution that stabilizes a helix by allowing for increased solvation of its N-terminus.
Co-reporter:Tingjian Chen, Floyd E. Romesberg
FEBS Letters (21 January 2014) Volume 588(Issue 2) pp:219-229
Publication Date(Web):21 January 2014
DOI:10.1016/j.febslet.2013.10.040
Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.
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