Krzysztof Kuczera

Name: Kuczera, Krzysztof

Organization: University of Kansas , USA

Department: Department of Molecular Biosciences

Title: Professor(PhD)

Chemicals
References

Finding optimal paths through biomolecular mazes: Comment on: “Ligand diffusion in proteins via enhanced sampling in molecular dynamics” by J. Rydzewski and W. Nowak

Co-reporter:Krzysztof Kuczera

Physics of Life Reviews 2017 Volumes 22–23(Volumes 22–23) pp:

Publication Date(Web):1 December 2017

DOI:10.1016/j.plrev.2017.08.001

Non-Exponential Kinetics and a Complete Folding Pathway of an α-Helical Heteropeptide: Direct Observation and Comprehensive Molecular Dynamics

Co-reporter:Gouri S. Jas, C. Russell Middaugh, and Krzysztof Kuczera

The Journal of Physical Chemistry B 2014 Volume 118(Issue 2) pp:639-647

Publication Date(Web):December 16, 2013

DOI:10.1021/jp410934g

We have performed a combined experimental and computational study of the folding of a 21-residue α-helix-forming heteropeptide (WH21). Temperature jump kinetics with improved dynamic range at several temperatures revealed non-exponential relaxation that could be well described with two time constants of 20 and 300 ns at 298 K. In the computational part, we performed multi-microsecond molecular dynamics simulations of WH21 in explicit water, using the AMBER03 and OPLS/AA potentials. The simulations were in good agreement with available experimental data on helix content and relaxation times. On the basis of 70 individual transitions, we identified the main pathways of helix unfolding. Three paths were found in both force fields, with unfolding progressing through (1) N-terminus, C-terminus, and center; (2) C-terminus, N-terminus, and center; and (3) C-terminus, center, and N-terminus. An additional fourth path starting in the central region and expanding to the termini was detected only with AMBER03. Intermediate structures sampled along the pathway included a central helix with frayed termini, an off-center helix, and a helical hairpin. The simulations suggest that the short relaxation should be assigned to partly cooperative fluctuations of several neighboring hydrogen bonds. Overall, by a combination of ultrafast kinetic measurements and detailed microscopic description through comprehensive molecular dynamics, we have obtained important new insights into the helix folding process.

Single Molecule Studies of Force-Induced S2 Site Exposure in the Mammalian Notch Negative Regulatory Domain

Co-reporter:Nicoleta Ploscariu, Krzysztof Kuczera, Katarzyna E. Malek, Magdalena Wawrzyniuk, Ashim Dey, and Robert Szoszkiewicz

The Journal of Physical Chemistry B 2014 Volume 118(Issue 18) pp:4761-4770

Publication Date(Web):April 15, 2014

DOI:10.1021/jp5004825

Notch signaling in metazoans is responsible for key cellular processes related to embryonic development and tissue homeostasis. Proteolitic cleavage of the S2 site within an extracellular NRR domain of Notch is a key early event in Notch signaling. We use single molecule force–extension (FX) atomic force microscopy (AFM) to study force-induced exposure of the S2 site in the NRR domain from mouse Notch 1. Our FX AFM measurements yield a histogram of N-to-C termini lengths, which we relate to conformational transitions within the NRR domain. We detect four classes of such conformational transitions. From our steered molecular dynamics (SMD) results, we associate first three classes of such events with the S2 site exposure. AFM experiments yield their mean unfolding forces as 69 ± 42, 79 ± 45, and 90 ± 50 pN, respectively, at 400 nm/s AFM pulling speeds. These forces are matched by the SMD results recalibrated to the AFM force loading rates. Next, we provide a conditional probability analysis of the AFM data to support the hypothesis that a whole sequence of conformational transitions within those three clases is the most probable pathway for the force-induced S2 site exposure. Our results support the hypothesis that force-induced Notch activation requires ligand binding to exert mechanical force not in random but in several strokes and over a substantial period of time.

Detailed Microscopic Unfolding Pathways of an α-Helix and a β-Hairpin: Direct Observation and Molecular Dynamics

Co-reporter:Gouri S. Jas, Wendy A. Hegefeld, C. Russell Middaugh, Carey K. Johnson, and Krzysztof Kuczera

The Journal of Physical Chemistry B 2014 Volume 118(Issue 26) pp:7233-7246

Publication Date(Web):June 4, 2014

DOI:10.1021/jp500955z

We present a combined experimental and computational study of unfolding pathways of a model 21-residue α-helical heteropeptide (W1H5-21) and a 16-residue β-hairpin (GB41–56). Experimentally, we measured fluorescence energy transfer efficiency as a function of temperature, employing natural tryptophans as donors and dansylated lysines as acceptors. Secondary structural analysis was performed with circular dichroism and Fourier transform infrared spectroscopy. Our studies present markedly different unfolding pathways of the two elementary secondary structural elements. During thermal denaturation, the helical peptide exhibits an initial decrease in length, followed by an increase, while the hairpin undergoes a systematic increase in length. In the complementary computational part of the project, we performed microsecond length replica-exchange molecular dynamics simulations of the peptides in explicit solvent, yielding a detailed microscopic picture of the unfolding processes. For the α-helical peptide, we found a large heterogeneous population of intermediates that are primarily frayed single helices or helix-turn-helix motifs. Unfolding starts at the termini and proceeds through a stable helical region in the interior of the peptide but shifted off-center toward the C-terminus. The simulations explain the experimentally observed non-monotonic variation of helix length with temperature as due primarily to the presence of frayed-end single-helix intermediate structures. For the β-hairpin peptide, our simulations indicate that folding is initiated at the turn, followed by formation of the hairpin in zipper-like fashion, with Cα···Cα contacts propagating from the turn to termini and hairpin hydrogen bonds forming in parallel with these contacts. In the early stages of hairpin formation, the hydrophobic side-chain contacts are only partly populated. Intermediate structures with low numbers of β-hairpin hydrogen bonds have very low populations. This is in accord with the “broken zipper” model of Scheraga. The monotonic increase in length with temperature may be explained by the zipper-like breaking of the hairpin hydrogen bonds and backbone contacts.

Influence of point mutations on the flexibility of cytochrome b5: Molecular dynamics simulations of holoproteins

Co-reporter:Qinyi Cheng;David R. Benson;Mario Rivera

Biopolymers 2006 Volume 83(Issue 3) pp:

Publication Date(Web):28 JUN 2006

DOI:10.1002/bip.20563

Two membrane-bound isoforms of cytochrome b₅ have been identified in mammals, one associated with the outer mitochondrial membrane (OM b₅) and the other with the endoplasmic reticulum (microsomal, or Mc b₅). The soluble heme binding domains of OM and Mc b₅ have highly similar three-dimensional structures but differ significantly in physical properties, with OM b₅ exhibiting higher stability due to stronger heme association. In this study, we present results of 8.5-ns length molecular dynamics simulations for rat Mc b₅, bovine Mc b₅, and rat OM b₅, as well as for two rat OM b₅ mutants that were anticipated to exhibit properties intermediate between those of rat OM b₅ and the two Mc proteins: the A18S/I32L/L47R triple mutant (OM^3M) and the A18S/I25L/I32L/L47R/L71S quintuple mutant (OM^5M). Analysis of the structure, fluctuations, and interactions showed that the five b₅ variants used in this study differed in organization of their molecular surfaces and heme binding cores in a way that could be used to explain certain experimentally observed physical differences. Overall, our simulations provided qualitative microscopic explanations of many of the differences in physical properties between OM and Mc b₅ and two mutants in terms of localized changes in structure and flexibility. They also reveal that opening of a surface cleft between hydrophobic cores 1 and 2 in bovine Mc b₅, observed in two previously reported simulations (E. M. Storch and V. Daggett, Biochemistry, 1995, Vol. 34, pp. 9682–9693; A. Altuve, Biochemistry, 2001, Vol. 40, pp. 9469–9483), probably resulted from removal of crystal contacts and likely does not occur on the nanosecond time scale. Finally, the MD simulations of OM^5M b₅ verify that stability and dynamic properties of cytochrome b₅ are remarkably resistant to mutations that dramatically alter the stability and structure of the apoprotein. © 2006 Wiley Periodicals, Inc. Biopolymers 83:297–312, 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

Structure, dynamics and interaction with kinase targets: computer simulations of calmodulin

Co-reporter:Cheng Yang, Gouri S. Jas, Krzysztof Kuczera

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2004 Volume 1697(1–2) pp:289-300

Publication Date(Web):11 March 2004

DOI:10.1016/j.bbapap.2003.11.032

Calmodulin (CaM) is a small protein involved in calcium signaling; among the targets of CaM are a number of kinases, including myosin light chain kinases (MLCK), various CaM-dependent kinases and phosphorylase kinase. We present results of molecular dynamics (MD) simulations of 4-ns length for calmodulin in its three functional forms: calcium-free, calcium-loaded, and in complex with both calcium and a target peptide, a fragment of the smooth muscle MLCK. The simulations included explicit water under realistic conditions of constant temperature and pressure, the presence of counterions and Ewald summation of electrostatic forces. Our simulation results present a more complete description of calmodulin structure, dynamics and interactions in solution than previously available. The results agree with a wide range of experimental data, including X-ray, nuclear magnetic resonance (NMR), fluorescence, cross-linking, mutagenesis and thermodynamics. Additionally, we are able to draw interesting conclusions about microscopic properties related to the protein's biological activity. First, in accord with fluorescence data, we find that calcium-free and calcium-loaded calmodulin exhibit significant structural flexibility. Our simulations indicate that these motions may be described as rigid-body translations and rotations of the N- and C-terminal domains occurring on a nanosecond time scale. Our second conclusion deals with the standard model of calmodulin action, which is that calcium binding leads to solvent exposure of hydrophobic patches in the two globular domains, which thus become ready to interact with the target. Surprisingly, the simulation results are inconsistent with the activation model when the standard definitions of the hydrophobic patches are used, based on hydrophobic clefts found in the X-ray structure of calcium-loaded calmodulin. We find that both experimental and simulation results are consistent with the activation model after a redefinition of the hydrophobic patches as those residues which are actually involved in peptide binding in the experimental structure of the calmodulin–peptide complex. The third conclusion is that the calmodulin–peptide interactions in the complex are very strong and are dominated by hydrophobic effects. Using quasi-harmonic entropy calculations, we find that these strong interactions induce a significant conformational strain in the protein and peptide. This destabilizing entropic contribution leads to a moderate overall binding free energy in the complex. Our results provide interesting insights into calmodulin binding to its kinase targets. The flexibility of the protein may explain the fact that CaM is able to bind many different targets. The large loss of conformational entropy upon CaM:peptide binding cancels the entropy gain due to hydrophobic interactions. This explains why the observed entropic contribution to the binding free energy is small and positive, and not large and negative as expected for a complex with such extensive hydrophobic contacts.

Molecular dynamics simulation studies of cytochrome b5 from outer mitochondrial and microsomal membrane

Co-reporter:Kyung-Hoon Lee

Biopolymers 2003 Volume 69(Issue 2) pp:

Publication Date(Web):15 APR 2003

DOI:10.1002/bip.10360

Two forms of cytochrome b₅ have been identified, associated with the outer membrane of liver mitochondria (OM cyt b₅) and with the membrane of the endoplasmic reticulum (microsomal, Mc cyt b₅). These proteins have very similar structures, but differ significantly in physical properties, with the OM cyt b₅ exhibiting a more negative reduction potential, higher stability, and stronger interactions with the heme. We perform molecular dynamics simulations to probe the structures and fluctuations of the two proteins in solution, to help explain the observed physical differences. We find that the structures of the two proteins, highly similar in the crystal, differ in position of a surface loop involving residues 49–51 in solution. Hydrophobic residues Ala-18, Ile-32, Leu-36, and Leu-47 tend to cluster together on the surface of rat OM cyt b₅, blocking water access to the protein interior. In bovine Mc cyt b₅, two of these positions, Ser-18 and Arg-47, are occupied by hydrophilic residues. This leads to breaking the hydrophobic cluster and allowing the protein to occupy a more open conformation. A measure of this structural transition is the opening of a cleft on the protein surface, which is 5 Å wider in the OM cyt b₅ simulation compared to the Mc form. The OM protein also appears to have a more compact hydrophobic core in its β-sheet region. These effects may be used to explain observed stability differences between the two proteins. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 260–269, 2003

Milestoning Simulation Reveals Mechanism of Helix-Breaking

Co-reporter:Krzysztof Kuczera

Biophysical Journal (20 August 2013) Volume 105(Issue 4) pp:

Publication Date(Web):20 August 2013

DOI:10.1016/j.bpj.2013.06.034