Co-reporter:Kalkena Sivanesam and Niels H. Andersen
Biochemistry October 10, 2017 Volume 56(Issue 40) pp:5373-5373
Publication Date(Web):September 18, 2017
DOI:10.1021/acs.biochem.7b00739
To date, fragments from within the amyloidogenic-patch region of human amylin (hAM) have been shown to aggregate independently of the full-length peptide. In this study, we show that under certain conditions, both oligomers of NFGAILSS and the monomeric form are capable of inhibiting the aggregation of the full-length hAM sequence. The inhibition, rather than aggregate seeding, observed with the soluble portion of aged NFGAILSS solutions was particularly striking occurring at far substoichiometric levels. Apparently, the oligomer form of this fragment is responsible for inhibiting the transition from random coil to β-sheet or serves as a disaggregator of hAM β-oligomers. Sequential deletion of the serine residues from NFGAILSS results in a decrease of inhibition, indicating that these residues are important to the activity of this fragment. We, like others, observed instances of α-helix-like CD spectra prior to β-sheet formation as part of the amyloidogenesis pathway. The partially aggregated sample and the fragments studied display spectroscopic diagnostics, suggesting that they slow down the conversion of full-length hAM monomers to cytotoxic oligomers.
Co-reporter:Jordan M. Anderson, Brice Jurban, Kelly N. L. Huggins, Alexander A. Shcherbakov, Irene Shu, Brandon Kier, and Niels H. Andersen
Biochemistry 2016 Volume 55(Issue 39) pp:5537
Publication Date(Web):September 7, 2016
DOI:10.1021/acs.biochem.6b00732
Many factors influence the stability of hairpins that could appear as foldons in partially folded states of proteins; of these, the propensity of certain amino acid sequences to favor conformations that serve to align potential β-strands for antiparallel association is likely the dominant feature. Quantitating turn propensities is viewed as the first step in developing an algorithm for locating nascent hairpins in protein sequences. Such nascent hairpins can serve to accelerate protein folding or, if they represent structural elements that differ from the final folded state, as kinetic traps. We have measured these “turn propensities” for the two most common turn types using a series of model peptide hairpins with four- and six-residue loops connecting the associated β-strands. Loops of four to six residues with specific turn sequences containing only natural l-amino acids and glycine can provide as much as 15 kJ/mol of hairpin stabilization versus loops lacking the defined turn loci. Single-site mutations within some of the optimal connecting loops can have ΔΔG effects as large as 9–10 kJ/mol on hairpin stability. In contrast to the near universal II′/I′ turns of model hairpins, a number of hairpin-supporting XZZG sequence β-turns with αR and/or γR configurations at the ZZ unit were found. A series of turn replacements (four-residue β-turns replaced by sequences that favor five- and six-residue reversing loops) using identical strands in our model systems have confirmed that several sequences have intrinsic turn propensities that could favor β-strand association in a non-native strand register and thus serve as kinetic traps. These studies also indicate that aryl residues immediately flanking a turn sequence can alter relative turn propensities by as much as 9–11 kJ/mol and will need to be a part of any nascent hairpin recognition algorithm.
Co-reporter:Jordan M. Anderson;Bron L. Kier;Brice Jurban;Aimee Byrne;Irene Shu;Lisa A. Eidenschink;Alexer A. Shcherbakov;Mike Hudson;R. M. Fesinmeyer
Biopolymers 2016 Volume 105( Issue 6) pp:337-356
Publication Date(Web):
DOI:10.1002/bip.22821
ABSTRACT
We have extended our studies of Trp/Trp to other Aryl/Aryl through-space interactions that stabilize hairpins and other small polypeptide folds. Herein we detail the NMR and CD spectroscopic features of these types of interactions. NMR data remains the best diagnostic for characterizing the common T-shape orientation. Designated as an edge-to-face (EtF or FtE) interaction, large ring current shifts are produced at the edge aryl ring hydrogens and, in most cases, large exciton couplets appear in the far UV circular dichroic (CD) spectrum. The preference for the face aryl in FtE clusters is W ≫ Y ≥ F (there are some exceptions in the Y/F order); this sequence corresponds to the order of fold stability enhancement and always predicts the amplitude of the lower energy feature of the exciton couplet in the CD spectrum. The CD spectra for FtE W/W, W/Y, Y/W, and Y/Y pairs all include an intense feature at 225–232 nm. An additional couplet feature seen for W/Y, W/F, Y/Y, and F/Y clusters, is a negative feature at 197–200 nm. Tyr/Tyr (as well as F/Y and F/F) interactions produce much smaller exciton couplet amplitudes. The Trp-cage fold was employed to search for the CD effects of other Trp/Trp and Trp/Tyr cluster geometries: several were identified. In this account, we provide additional examples of the application of cross-strand aryl/aryl clusters for the design of stable β-sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 337–356, 2016.
Co-reporter:Brandon L. Kier; Jordan M. Anderson
Journal of the American Chemical Society 2015 Volume 137(Issue 16) pp:5363-5371
Publication Date(Web):April 2, 2015
DOI:10.1021/ja5117809
Disulfide bonds between cysteine residues are essential to the structure and folding of many proteins. Yet their role in the design of structured peptides and proteins has frequently been limited to use as intrachain covalent staples that reinforce existing structure or induce knot-like conformations. In β-hairpins, their placement at non-H-bonding positions across antiparallel strands has proven useful for achieving fully folded positive controls. Here we report a new class of designed β-sheet peptide dimers with strand-central disulfides as a key element. We have found that the mere presence of a disulfide bond near the middle of a short peptide chain is sufficient to nucleate some antiparallel β-sheet structure; addition of β-capping units and other favorable cross-strand interactions yield hyperstable sheets. Strand-central cystines were found to be superior to the best designed reversing turns in terms of nucleating β-sheet structure formation. We have explored the limitations and possibilities of this technique (the use of disulfides as sheet nucleators), and we provide a set of rules and rationales for the application and further design of disulfide-tethered “turnless” β-sheets.
Co-reporter:K. Sivanesam, A. Byrne, M. Bisaglia, L. Bubacco and N. Andersen
RSC Advances 2015 vol. 5(Issue 15) pp:11577-11590
Publication Date(Web):16 Jan 2015
DOI:10.1039/C5RA00325C
Further examination of peptides with well-folded antiparallel β strands as inhibitors of amyloid formation from α-synuclein has resulted in more potent inhibitors. Several of these had multiple Tyr residues and represent a new lead for inhibitor design by small peptides that do not divert α-synuclein to non-amyloid aggregate formation. The most potent inhibitor obtained in this study is a backbone cyclized version of a previously studied β hairpin, designated as WW2, with a cross-strand Trp/Trp cluster. The cyclization was accomplished by adding a D-Pro-L-Pro turn locus across strand termini. At a 2:1 peptide to α-synuclein ratio, cyclo-WW2 displays complete inhibition of β-structure formation. Trp-bearing antiparallel β-sheets held together by a disulphide bond are also potent inhibitors. 15N HSQC spectra of α-synuclein provided new mechanistic details. The time course of 15N HSQC spectral changes observed during β-oligomer formation has revealed which segments of the structure become part of the rigid core of an oligomer at early stages of amyloidogenesis and that the C-terminus remains fully flexible throughout the process. All of the effective peptide inhibitors display binding-associated titration shifts in 15N HSQC spectra of α-synuclein in the C-terminal Q109-E137 segment. Cyclo-WW2, the most potent inhibitor, also displays titration shifts in the G41–T54 span of α-synuclein, an additional binding site. The earliest aggregation event appears to be centered about H50 which is also a binding site for our most potent inhibitor.
Co-reporter:Aimee Byrne, D. Victoria Williams, Bipasha Barua, Stephen J. Hagen, Brandon L. Kier, and Niels H. Andersen
Biochemistry 2014 Volume 53(Issue 38) pp:
Publication Date(Web):September 3, 2014
DOI:10.1021/bi501021r
Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9–7.5 μs at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.
Co-reporter:Brandon L. Kier ; Jordan M. Anderson
Journal of the American Chemical Society 2013 Volume 136(Issue 2) pp:741-749
Publication Date(Web):December 18, 2013
DOI:10.1021/ja410824x
A hyperstable Pin1 WW domain has been circularly permuted via excision of the fold-nucleating turn; it still folds to form the native three-strand sheet and hydrophobic core features. Multiprobe folding dynamics studies of the normal and circularly permuted sequences, as well as their constituent hairpin fragments and comparable-length β-strand-loop-β-strand models, indicate 2-state folding for all topologies. N-terminal hairpin formation is the fold nucleating event for the wild-type sequence; the slower folding circular permutant has a more distributed folding transition state.
Co-reporter:Aimee Byrne, Brandon L. Kier, D. V. Williams, Michele Scian and Niels H. Andersen
RSC Advances 2013 vol. 3(Issue 43) pp:19824-19829
Publication Date(Web):02 Sep 2013
DOI:10.1039/C3RA43674H
The Trp-cage, at 20 residues in length, is generally acknowledged as the smallest fully protein-like folding motif. Linking the termini by a two-residue unit and excising one residue affords circularly permuted sequences that adopt the same structure. This represents the first successful circular permutation of any fold of less than 50-residue length. As was observed for the original topology, a hydrophobic staple near the chain termini is required for enhanced fold stability.
Co-reporter:Michele Scian, Irene Shu, Katherine A. Olsen, Khalil Hassam, and Niels H. Andersen
Biochemistry 2013 Volume 52(Issue 15) pp:
Publication Date(Web):March 22, 2013
DOI:10.1021/bi400146c
The fold stabilities and folding dynamics of a series of mutants of a model hairpin, KTW-NPATGK-WTE (HP7), are reported. The parent system and the corresponding DPATGK loop species display submicrosecond folding time constants. The mutational studies revealed that ultrafast folding requires both some prestructuring of the loop and a favorable interaction between the chain termini in the transition state. In the case of YY-DPETGT-WY, another submicrosecond folding species [Davis, C. M., Xiao, S., Raleigh, D. P., and Dyer, R. B. (2012) J. Am. Chem. Soc. 134, 14476–14482], a hydrophobic cluster provides the latter. In the case of HP7, the Coulombic interaction between the terminal NH3+ and CO2– units provides this; a C-terminal Glu to amidated Ala mutation results in a 5-fold retardation of the folding rate. The effects of mutations within the reversing loop indicate the balance between loop flexibility (favoring fast conformational searching) and turn formation in the unfolded state is a major factor in determining the folding dynamics. The -NAAAKX- loops examined display no detectable turn formation propensity in other hairpin constructs but do result in stable analogues of HP7. Peptide KTW-NAAAKK-WTE displays the same fold stability as HP7, but both the folding and unfolding time constants are greater by a factor of 20.
Co-reporter:Michele Scian;George I. Makhatadze;Isolde Le Trong;Jasper C. Lin;Ronald E. Stenkamp
PNAS 2012 Volume 109 (Issue 31 ) pp:
Publication Date(Web):2012-07-31
DOI:10.1073/pnas.1121421109
To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version
of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting
higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations
that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated
with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior. A hexameric
oligomer of the cyclic peptide is found in both crystal forms and indicates that under appropriate conditions, this minimized
system may also serve as a model for protein–protein interactions.
Co-reporter:Irene Shu ; James M. Stewart ; Michele Scian ; Brandon L. Kier
Journal of the American Chemical Society 2011 Volume 133(Issue 5) pp:1196-1199
Publication Date(Web):January 7, 2011
DOI:10.1021/ja1088953
The 13C chemical shifts measured for designed β-hairpins indicate that the structuring shifts (upfield for Cα and C′, downfield for Cβ) previously reported as diagnostic for β-structuring in proteins appear only at the H-bonded strand residues. The resulting periodicity of structuring shift magnitudes is not, however, a consequence of H-bonding status; rather, it reflects a previously unrecognized alternation in the backbone torsion angles of β-strands. This feature of hairpins is also likely to be present in proteins. The study provides reference values for the expectation shifts for 13C sites in β-structures that should prove useful in the characterization of the folding equilibria of β-sheet models.
Co-reporter:
Biochemistry 2011 Volume 50(Issue 7) pp:1143-1152
Publication Date(Web):January 11, 2011
DOI:10.1021/bi101555y
Gai and co-workers [Bunagan, M. R., et al. (2006) J. Phys. Chem. B 110, 3759−3763] reported computational design studies suggesting that a D9E mutation would stabilize the Trp-cage. Experimental studies for this mutation were reported in 2008 [Hudaky, P., et al. (2008) Biochemistry 47, 1007−1016]; the authors suggested that [D9E]-TC5b presented a more compact and melting resistant structure because of the “optimal distance between the two sides of the molecule”. Nonetheless, the authors reported essentially the same circular dichroism (CD) melting temperature, 38 ± 0.3 °C, for TC5b and its [D9E] mutant. In this study, a more stable Trp-cage, DAYAQ WLKDG GPSSG RPPPS, was examined by nuclear magnetic resonance and CD with the following mutations: [D9E], [D9R,R16E], [R16O], [D9E,R16O], [R16K], and [D9E,R16K]. Of these, the [D9E] mutant displayed the smallest acidification-induced change in the apparent Tm. In analogy to the prior study, the CD melts of TC10b and its [D9E] mutant were, however, very similar; all of the other mutations were significantly fold destabilizing by all measures. A detailed analysis indicates that the original D9−R16 salt bridge is optimal with regard to fold cooperativity and fold stabilization. Evidence of salt bridge formation is also provided for a swapped pair, the [D9R,R16E] mutant. Model systems reveal that an ionized aspartate at the C-terminus of a helix significantly decreases intrinsic helicity, a requirement for Trp-cage fold stability. The CD evidence that was cited as supporting increased fold stability for [D9E]-TC5b at higher temperatures appears to be a reflection of increased helix stability in both the folded and unfolded states rather than a more favorable salt bridge. Our study also provides evidence of other Trp-cage stabilizing roles of the R16 side chain.
Co-reporter:Kelly N. L. Huggins, Marco Bisaglia, Luigi Bubacco, Marianna Tatarek-Nossol, Aphrodite Kapurniotu, and Niels H. Andersen
Biochemistry 2011 Volume 50(Issue 38) pp:
Publication Date(Web):August 17, 2011
DOI:10.1021/bi200760h
Hairpin peptides bearing cross-strand Trp-Trp and Tyr-Tyr pairs at non-H-bonded strand sites modulate the aggregation of two unrelated amyloidogenic systems, human pancreatic amylin (hAM) and α-synuclein (α-syn), associated with type II diabetes and Parkinson’s disease, respectively. In the case of hAM, we have previously reported that inhibition of amyloidogenesis is observed as an increase in the lag time to amyloid formation and a diminished thioflavin (ThT) fluorescence response. In this study, a reduced level of hAM fibril formation is confirmed by transmission electron microscopy imaging. Several of the hairpins tested were significantly more effective inhibitors than rat amylin. Moreover, a marked inhibitory effect on hAM-associated cytotoxicity by the more potent hairpin peptide is demonstrated. In the case of α-syn, the dominant effect of active hairpins was, besides a weakened ThT fluorescence response, the earlier appearance of insoluble aggregates that do not display amyloid characteristics with the few fibrils observed having abnormal morphology. We attribute the alteration of the α-synuclein aggregation pathway observed to the capture of a preamyloid state and diversion to nonamyloidogenic aggregates. These β-hairpins represent a new class of amyloid inhibitors that bear no sequence similarity to the amyloid-producing polypeptides that are inhibited. A mechanistic rationale for these effects is proposed.
Co-reporter:Irene Shu;Lisa A. Eidenschink;Brandon L. Kier
PNAS 2010 Volume 107 (Issue 23 ) pp:10466-10471
Publication Date(Web):2010-06-08
DOI:10.1073/pnas.0913534107
Although much has been learned about the design of models of β-sheets during the last decade, modest fold stabilities in water
and terminal fraying remain a feature of most β-hairpin peptides. In the case of hairpin capping, nature did not provide guidance
for solving the problem. Some observations from prior turn capping designs, with further optimization, have provided a generally
applicable, “unnatural” beta cap motif (alkanoyl-Trp at the N terminus and Trp-Thr-Gly at the C terminus) that provides a
net contribution of 6 + kJ/mol to β-hairpin stability, surpassing all other interactions that stabilize β-hairpins including
the covalent disulfide bond. The motif, made up entirely of natural residues, is specific to the termini of antiparallel β-strands
and reduces fraying at the ends of hairpins and other β-sheet models. Utilizing this motif, 10- to 22-residue peptide scaffolds
of defined stereochemistry that are greater than 98% folded in water have been prepared. The β-cap can also be used to staple
together short antiparallel β-strands connected by a long flexible loop.
Co-reporter:Lisa Eidenschink;Edward Crabbe
Biopolymers 2009 Volume 91( Issue 7) pp:557-564
Publication Date(Web):
DOI:10.1002/bip.21177
Abstract
While end capping in α-helices is well understood, the concept of capping a β-hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β-hairpin. While cross-strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β-hairpin a conservative, hydrophobic, V I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H-bonded site and the sidechain at the C-terminal non-H-bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C-terminal site, for example an E A mutation (ΔΔGU = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009.
This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Co-reporter:Brandon L. Kier
Journal of the American Chemical Society 2008 Volume 130(Issue 44) pp:14675-14683
Publication Date(Web):October 9, 2008
DOI:10.1021/ja804656h
Mutational optimization of two long-range interactions first observed in Ac-WINGKWT-NH2, (a) bifurcated H-bonding involving the threonine amide HN and side chain OH and the N-terminal acetyl carbonyl and (b) an H-bond between the entgegen-HN of the C-terminal amide and the indole ring of Trp6 that stabilizes a face-to-edge indole/indole interaction between Trp1 and Trp6, has afforded ≤10 residue systems that yield a remarkably stable fold in water. Optimization was achieved by designing a hydrophobic cluster that sequesters these H-bonds from solvent exposure. The structures and extent of amide H/D exchange protection for CH3CH2CO-WIpGXWTGPS (p = d-Pro, X = Leu or Ile) were determined. These two systems are greater than 94% folded at 298 K (97.5% at 280 K) with melting temperatures >75 °C. The fold appears to display minimal fluxionality; a well-converged NMR structure rationalizes all of the large structuring shifts observed, and we suggest that these designed constructs can be viewed as microproteins.
Co-reporter:James M. Stewart, Jasper C. Lin and Niels H. Andersen
Chemical Communications 2008 (Issue 39) pp:4765-4767
Publication Date(Web):08 Aug 2008
DOI:10.1039/B807101B
The helix-disfavoring, versusalanine, propagation values of lysine (0.8) and arginine (1.0) residues placed centrally in an (Ala)9 unit have been measured by 13C NMR.
Co-reporter:D. Victoria Williams, Bipasha Barua and Niels H. Andersen
Organic & Biomolecular Chemistry 2008 vol. 6(Issue 23) pp:4287-4289
Publication Date(Web):15 Oct 2008
DOI:10.1039/B814314E
The folding enantioselectivity for D-AlaversusL-Ala at one glycine site in the Trp-cage is 16 kJ mol−1; judicious introductions of alanines of the correct chirality raises the melting temperature of this 20-residue fold to 83 °C.
Co-reporter:Kun Song;James M. Stewart;R. Matthew Fesinmeyer;Carlos Simmerling
Biopolymers 2008 Volume 89( Issue 9) pp:747-760
Publication Date(Web):
DOI:10.1002/bip.21004
Abstract
The temperature dependence of helical propensities for the peptides Ac-ZGG-(KAAAA)3X-NH2 (Z = Y or G, X = A, K, and D-Arg) were studied both experimentally and by MD simulations. Good agreement is observed in both the absolute helical propensities as well as relative helical content along the sequence; the global minimum on the calculated free energy landscape corresponds to a single α-helical conformation running from K4 to A18 with some terminal fraying, particularly at the C-terminus. Energy component analysis shows that the single helix state has favorable intramolecular electrostatic energy due to hydrogen bonds, and that less-favorable two-helix globular states have favorable solvation energy. The central lysine residues do not appear to increase helicity; however, both experimental and simulation studies show increasing helicity in the series X = Ala Lys D-Arg. This C-capping preference was also experimentally confirmed in Ac-(KAAAA)3X-GY-NH2 and (KAAAA)3X-GY-NH2 sequences. The roles of the C-capping groups, and of lysines throughout the sequence, in the MD-derived ensembles are analyzed in detail. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 747–760, 2008.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Co-reporter:F. Michael Hudson
Biopolymers 2006 Volume 83(Issue 4) pp:
Publication Date(Web):18 JUL 2006
DOI:10.1002/bip.20575
Recently validated chemical shift measures of hairpin structuring have been applied to a series of turn mutants of the Schenck–Gellman three-strand β-sheet model with the aim of measuring the entropic advantage associated with aligning an additional strand onto an existing hairpin versus aligning the same two strands in an initial hairpin formation. In a four-state analysis (unfolded, 2 single hairpins, and the double hairpin fold in equilibrium) a cooperativity index can be defined as the factor by which the equilibrium constant for hairpin formation is improved when one strand is prestructured. This cooperativity index is 2.7 ± 0.7 for hairpin formation about a stable D-Pro-Gly turn locus and increases to 7.6 ± 1.2 for an Asn-Gly turn locus. The latter corresponds to a cooperativity induced ΔΔG increment of 4.9 kJ/mol for the folding of a hairpin. Although larger than previous experimental measures of folding cooperativity in three-stranded sheets, the magnitude of this effect (which is considerably less than the TΔΔS expectation for prestructuring three or more β-strand residue sites) likely reflects the intrinsic preference of these designed sequences for extended conformations. If similar or larger effects apply to protein β-sheet folding, it is not surprising that particularly favorable hairpin alignments serve as nucleation sites in protein folding pathways. © 2006 Wiley Periodicals, Inc. Biopolymers 83:424–433, 2006
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Co-reporter:Katherine A. Olsen;R. Matthew Fesinmeyer;James M. Stewart
PNAS 2005 102 (43 ) pp:15483-15487
Publication Date(Web):2005-10-25
DOI:10.1073/pnas.0504392102
Hairpins play a central role in numerous protein folding and misfolding scenarios. Prior studies of hairpin folding, many
conducted with analogs of a sequence from the B1 domain of protein G, suggest that faster folding can be achieved only by
optimizing the turn propensity of the reversing loop. Based on studies using dynamic NMR, the native GB1 sequence is a slow
folding hairpin . GB1 hairpin analogs spanning a wide range of thermodynamic stabilities were examined. Fold-stabilizing changes in the reversing loop can act either by accelerating folding or retarding unfolding;
we present examples of both types. The introduction of an attractive side-chain/side-chain Coulombic interaction at the chain
termini further stabilizes this hairpin. The 1.9-fold increase in folding rate constant observed for this change at the chain
termini implies that this Coulombic interaction contributes before or at the transition state. This observation is difficult
to rationalize by “zipper” folding pathways that require native turn formation as the sole nucleating event; it also suggests
that Coulombic interactions should be considered in the design of systems intended to probe the protein folding speed limit.
Co-reporter:Niels H Andersen, Yan Brodsky, Jonathan W Neidigh, Kathryn S Prickett
Bioorganic & Medicinal Chemistry 2002 Volume 10(Issue 1) pp:79-85
Publication Date(Web):January 2002
DOI:10.1016/S0968-0896(01)00263-2
Exendin-4 is a natural, 39-residue peptide first isolated from the salivary secretions of a Gila Monster (Heloderma suspectum) that has some pharmacological properties similar to glucagon-like-peptide-1 (GLP-1). This paper reports differences in the structural preferences of these two peptides. For GLP-1 in aqueous buffer (pH 3.5 or 5.9), the concentration dependence of circular dichroism spectra suggests that substantial helicity results only as a consequence of helix bundle formation. In contrast, exendin-4 is significantly helical in aqueous buffer even at the lowest concentration examined (2.3 μM). The pH dependence of the helical signal for exendin-4 indicates that helicity is enhanced by a more favorable sequence alignment of oppositely charged sidechains. Both peptides become more helical upon addition of either lipid micelles or fluoroalcohols. The stabilities of the helices were assessed from the thermal gradient of ellipticity (∂[θ]221/∂T values); on this basis, the exendin helix does not melt appreciably until temperatures significantly above ambient. The extent of helix formation for exendin-4 in aqueous buffer (and the thermal stability of the resulting helix) suggests the presence of a stable helix-capping interaction which was localized to the C-terminal segment by NMR studies of NH exchange protection. Solvent effects on the thermal stability of the helix indicate that the C-terminal capping interaction is hydrophobic in nature. The absence of this C-capping interaction and the presence of a flexible, helix-destabilizing glycine at residue 16 in GLP-1 are the likely causes of the greater fragility of the monomeric helical state of GLP-1. The intramolecular hydrophobic clustering in exendin-4 also appears to decrease the extent of helical aggregate formation.Exendin-4 is a 39-residue peptide from the salivary secretions of Heloderma suspectum having some pharmacological properties similar to GLP-1. The synthetic version of this peptide (AC2993) is currently in phase 2 clinical trials as a treatment as a treatment for type 2 diabetes. In contrast to GLP-1, exendin-4 forms monomeric helices in aqueous media. This helical state displays unusual thermal stability due to a hydrophobic C-terminal capping interaction. The absence of this C-capping interaction and the presence of a flexible, helix-destabilizing glycine at residue 16 in GLP-1 are proposed to be the basis for the diminished stability of the monomeric helical state of GLP-1. The greater intrinsic stability of the exendin-4 helix likely reduces the entropic cost of binding at a receptor that requires the C-terminal helix state.
Co-reporter:
Nature Structural and Molecular Biology 2002 9(6) pp:425 - 430
Publication Date(Web):29 April 2002
DOI:10.1038/nsb798
Co-reporter:Jonathan W. Neidigh
Biopolymers 2002 Volume 65(Issue 5) pp:
Publication Date(Web):10 OCT 2002
DOI:10.1002/bip.10272
Sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles are often used to mimic the membrane- or receptor-bound states of peptides in NMR studies. From the present examination of a 26-residue analog of exendin-4 (TrEX4) by NMR and CD in water, aqueous 30% trifluoroethanol (TFE), and bound to both SDS and DPC micelles, it is clear that these two lipid micelles can yield very different peptide structures. The Trp-cage fold (also observed in 30% TFE) is present when TrEX4 is bound to SDS micelles; however, tertiary structure is absent in the presence of DPC micelles. The loss of tertiary structure is attributed to an energetically favorable interaction (estimated as 2–3 kcal/mol) of the tryptophan side chain with the phosphocholine head groups. These dramatic structural differences suggest that care must be taken when using either SDS or DPC to mimic the membrane- or receptor-bound states. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 354–361, 2002
Co-reporter:James M. Stewart, Jasper C. Lin and Niels H. Andersen
Chemical Communications 2008(Issue 39) pp:
Publication Date(Web):
DOI:10.1039/B807101B
Co-reporter:D. Victoria Williams, Bipasha Barua and Niels H. Andersen
Organic & Biomolecular Chemistry 2008 - vol. 6(Issue 23) pp:NaN4289-4289
Publication Date(Web):2008/10/15
DOI:10.1039/B814314E
The folding enantioselectivity for D-AlaversusL-Ala at one glycine site in the Trp-cage is 16 kJ mol−1; judicious introductions of alanines of the correct chirality raises the melting temperature of this 20-residue fold to 83 °C.
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AMMONIO}ACETATE](http://img.cochemist.com/ccimg/38800/38754-71-1.png)
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