The mechanism of collagenolysis : a substrate-centric view

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Physics, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references.Collagenolysis (collagen degradation) is a physiological process involved in normal tissue maintenance, but excessive collagenolysis has been associated with the progression of cancer metastasis, atherosclerosis, and other diseases. Despite considerable efforts to understand the steps involved, the exact mechanism of collagenolysis remains unknown. One proposed mechanism suggests that the enzymes that degrade collagen, collagenases, physically unwind the triple-helical structure of collagen to gain access to the peptide bond that is cleaved. This unwinding mechanism would in principle have large energetic requirements, but neither ATP nor other energy-rich molecules are necessary for collagenolysis. An alternative mechanism is that collagen exists in multiple states, some featuring structures that are unfolded in the vicinity of the collagenase cleavage site, and that collagenases preferentially bind to and stabilize these partially unfolded structures before degradation occurs. The focus of this work is to investigate this alternative mechanism, particularly as it pertains to the conformational ensemble of collagen, using both experimental and computational methods. In particular, this work concentrates on: (1) devising a reaction scheme for this mechanism and verifying that it can explain existing experimental observations; (2) generating a structural model of the type I collagen collagenase cleavage site using molecular dynamics simulations; (3) validating this structural model by performing degradation experiments and analyzing them in light of the reaction scheme described in (1); and (4) validating a similar structural model for type III collagen by designing and characterizing a self-assembling collagen-like model peptide that includes the sequence of the collagenase cleavage site. Together, these data present a detailed and comprehensive analysis of the conformational ensemble of collagen near the collagenase cleavage site and its role in the molecular mechanism of collagen degradation.by Paul S. Nerenberg.Ph.D

Disordered Structural Ensembles of Vasopressin and Oxytocin and Their Mutants

Vasopressin and oxytocin are intrinsically disordered cyclic nonapeptides belonging to a family of neurohypophysial hormones. Although unique in their functions, these peptides differ only by two residues and both feature a tocin ring formed by the disulfide bridge between first and sixth cysteine residues. This sequence and structural similarity are experimentally linked to oxytocin agonism at vasopressin receptors and vasopressin antagonism at oxytocin receptors. Yet single- or double-residue mutations in both peptides have been shown to have drastic impacts on their activities at either receptor, and possibly the ability to bind to their neurophysin carrier protein. In this study we perform molecular dynamics simulations of the unbound native and mutant sequences of the oxytocin and vasopressin hormones to characterize their structural ensembles. We classify the subpopulations of these structural ensembles on the basis of the distributions of radius of gyration and secondary structure and hydrogen-bonding features of the canonical tocin ring and disordered tail region. We then relate the structural changes observed in the unbound form of the different hormone sequences to experimental information about peptide receptor binding, and more indirectly, carrier protein binding affinity, receptor activity, and protease degradation. This study supports the hypothesis that the structural characteristics of the unbound form of an IDP can be used to predict structural or functional preferences of its functional bound form

A practical guide to the simultaneous determination of protein structure and dynamics using metainference

Accurate protein structural ensembles can be determined with metainference, a Bayesian inference method that integrates experimental information with prior knowledge of the system and deals with all sources of uncertainty and errors as well as with system heterogeneity. Furthermore, metainference can be implemented using the metadynamics approach, which enables the computational study of complex biological systems requiring extensive conformational sampling. In this chapter, we provide a step-by-step guide to perform and analyse metadynamic metainference simulations using the ISDB module of the open-source PLUMED library, as well as a series of practical tips to avoid common mistakes. Specifically, we will guide the reader in the process of learning how to model the structural ensemble of a small disordered peptide by combining state-of-the-art molecular mechanics force fields with nuclear magnetic resonance data, including chemical shifts, scalar couplings and residual dipolar couplings.Comment: 49 pages, 9 figure

Identifying Alternative Hyper-Splicing Signatures in MG-Thymoma by Exon Arrays

BACKGROUND: The vast majority of human genes (>70%) are alternatively spliced. Although alternative pre-mRNA processing is modified in multiple tumors, alternative hyper-splicing signatures specific to particular tumor types are still lacking. Here, we report the use of Affymetrix Human Exon Arrays to spot hyper-splicing events characteristic of myasthenia gravis (MG)-thymoma, thymic tumors which develop in patients with MG and discriminate them from colon cancer changes. METHODOLOGY/PRINCIPAL FINDINGS: We combined GO term to parent threshold-based and threshold-independent ad-hoc functional statistics with in-depth analysis of key modified transcripts to highlight various exon-specific changes. These denote alternative splicing in MG-thymoma tumors compared to healthy human thymus and to in-house and Affymetrix datasets from colon cancer and healthy tissues. By using both global and specific, term-to-parent Gene Ontology (GO) statistical comparisons, our functional integrative ad-hoc method allowed the detection of disease-relevant splicing events. CONCLUSIONS/SIGNIFICANCE: Hyper-spliced transcripts spanned several categories, including the tumorogenic ERBB4 tyrosine kinase receptor and the connective tissue growth factor CTGF, as well as the immune function-related histocompatibility gene HLA-DRB1 and interleukin (IL)19, two muscle-specific collagens and one myosin heavy chain gene; intriguingly, a putative new exon was discovered in the MG-involved acetylcholinesterase ACHE gene. Corresponding changes in spliceosome composition were indicated by co-decreases in the splicing factors ASF/SF(2) and SC35. Parallel tumor-associated changes occurred in colon cancer as well, but the majority of the apparent hyper-splicing events were particular to MG-thymoma and could be validated by Fluorescent In-Situ Hybridization (FISH), Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and mass spectrometry (MS) followed by peptide sequencing. Our findings demonstrate a particular alternative hyper-splicing signature for transcripts over-expressed in MG-thymoma, supporting the hypothesis that alternative hyper-splicing contributes to shaping the biological functions of these and other specialized tumors and opening new venues for the development of diagnosis and treatment approaches

New developments in force fields for biomolecular simulations.

Biomolecular force field development has been instrumental in improving the predictive power of molecular simulations over the past four decades. More recently, the era of large quantitative experimental datasets and ubiquitous high performance computing power has enabled rapid progress in the field. In this review we summarize recent developments in all-atom protein, nucleic acid, and small molecule force fields, paying specific attention to developments in parameterization methods and improvements in the representations of nonbonded interactions that are critical for solving the challenging biophysical problems of the present. We also sketch out new avenues for force field development and grand challenge applications for the near future

Optimizing Protein-Solvent Force Fields to Reproduce Intrinsic Conformational Preferences of Model Peptides.

While most force field efforts in biomolecular simulation have focused on the parametrization of the protein, relatively little attention has been paid to the quality of the accompanying solvent model. These considerations are especially relevant for simulations of intrinsically disordered peptides and proteins, for which energy differences between conformations are small and interactions with water are enhanced. In this work, we investigate the accuracy of the AMBER ff99SB force field when combined with the standard TIP3P model or the more recent TIP4P-Ew water model, to generate conformational ensembles for disordered trialanine (Ala3), triglycine (Gly3), and trivaline (Val3) peptides. We find that the TIP4P-Ew water model yields significantly better agreement with experimentally measured scalar couplings-and therefore more accurate conformational ensembles-for both Ala3 and Gly3. For Val3, however, we find that the TIP3P and TIP4P-Ew ensembles are equivalent in their performance. To further improve the protein-water force field combination and obtain more accurate intrinsic conformational preferences, we derive a straightforward perturbation to the ϕ' backbone dihedral potential that shifts the β-PPII equilibrium. We find that the revised ϕ' backbone dihedral potential yields improved conformational ensembles for a variety of small peptides and maintains the stability of the globular ubiquitin protein in TIP4P-Ew water

Optimizing Protein-Solvent Force Fields to Reproduce Intrinsic Conformational Preferences of Model Peptides.

While most force field efforts in biomolecular simulation have focused on the parametrization of the protein, relatively little attention has been paid to the quality of the accompanying solvent model. These considerations are especially relevant for simulations of intrinsically disordered peptides and proteins, for which energy differences between conformations are small and interactions with water are enhanced. In this work, we investigate the accuracy of the AMBER ff99SB force field when combined with the standard TIP3P model or the more recent TIP4P-Ew water model, to generate conformational ensembles for disordered trialanine (Ala3), triglycine (Gly3), and trivaline (Val3) peptides. We find that the TIP4P-Ew water model yields significantly better agreement with experimentally measured scalar couplings-and therefore more accurate conformational ensembles-for both Ala3 and Gly3. For Val3, however, we find that the TIP3P and TIP4P-Ew ensembles are equivalent in their performance. To further improve the protein-water force field combination and obtain more accurate intrinsic conformational preferences, we derive a straightforward perturbation to the ϕ' backbone dihedral potential that shifts the β-PPII equilibrium. We find that the revised ϕ' backbone dihedral potential yields improved conformational ensembles for a variety of small peptides and maintains the stability of the globular ubiquitin protein in TIP4P-Ew water

The Contribution of Interchain Salt Bridges to Triple-Helical Stability in Collagen

Studies on collagen and collagen-like peptides suggest that triple-helical stability can vary along the amino acid chain. In this regard, it has been shown that lysine residues in the Y position and acidic residues in the X′ position of (GPO)3GXYGX′Y′(GPO)3 peptides lead to triple-helical structures with melting temperatures similar to (GPO)8 (where O is hydroxyproline), which is generally regarded as the most stable collagen-like sequence of this length. This enhanced stability has been attributed to the formation of salt bridges between adjacent collagen chains. In this study, we explore the relationship between interchain salt bridge formation and triple-helical stability using detailed molecular simulations. Although our results confirm that salt bridges promote triple-helical stability, we find that not all salt bridges are created equal. In particular, lysine-glutamate salt bridges are most stabilizing when formed between residues in the middle strand (B) and the trailing strand (C), whereas lysine-aspartate salt bridges are most stabilizing when formed between residues in the leading (A) and middle (B) strand—the latter observation being consistent with recent NMR data on a heterotrimeric model peptide. Overall, we believe these data clarify the role of salt bridges in modulating triple-helical stability and can be used to guide the design of collagen-like peptides that have specific interchain interactions