The role of stabilization centers in protein thermal stability

AbstractThe definition of stabilization centers was introduced almost two decades ago. They are centers of noncovalent long range interaction clusters, believed to have a role in maintaining the three-dimensional structure of proteins by preventing their decay due to their cooperative long range interactions. Here, this hypothesis is investigated from the viewpoint of thermal stability for the first time, using a large protein thermodynamics database. The positions of amino acids belonging to stabilization centers are correlated with available experimental thermodynamic data on protein thermal stability. Our analysis suggests that stabilization centers, especially solvent exposed ones, do contribute to the thermal stabilization of proteins

DNA-Mediated Electrochemistry

The base pair stack of DNA has been demonstrated as a medium for long-range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here, we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry

Four Levels of Hierarchical Organization, Including Noncovalent Chainmail, Brace the Mature Tumor Herpesvirus Capsid against Pressurization

SummaryLike many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization

Methods and Informatics for Gas-Phase Structural Biology and Drug Discovery

Methods for rapid interrogation of structure and stability attributes of proteins and protein complexes are becoming increasingly important for developing our understanding of biology and the development of pharmaceuticals. Gas-phase technologies such as mass spectrometry and ion mobility spectrometry have proven valuable in these endeavors, as they provide unique perspectives on the solution-phase equilibrium of protein complexes and their conformations. Before fully harnessing the information derived from these gas-phase techniques, new approaches for data analysis and mechanistic understanding of gas-phase protein structure are necessary. In this dissertation, we develop ion mobility mass spectrometry methods and informatics for the study of gas-phase proteins, multiprotein complexes, and protein-small molecule complexes. In the first half of the dissertation, novel data analysis tools and experimental methodologies are outlined for the study of gas-phase protein unfolding. After providing the software tools necessary for robust analysis of gas-phase unfolding trajectories in Chapter 2, we turned our attention to understanding the mechanism of unfolding for large multidomain proteins. In Chapter 3, we focus on the factors driving changes in unfolding trajectories for a variety of serum albumin homologues, and through the use of novel unfolding experiments utilizing chemical probes and non-covalent protein constructs, a detailed mechanism for solvent-free protein unfolding is provided. Subsequent chapters in the dissertation focus on the characterization of multiprotein complexes, especially through the use of ion mobility-mass spectrometry and coarse-grained modeling. In chapter 4, we develop and benchmark new algorithms for translating ion mobility and mass spectrometry datasets into coarse grained models. These studies outline the limits in current coarse-graining methodologies, and define the minimum restraint sets necessary to generate high confidence multiprotein models. Additionally, best practices for dealing with ambiguous models resulting from sparse datasets are described. In chapter 5, the tools developed in the previous chapter are applied to structurally characterize the urease pre-activation complex, a transient 18-subunit complex that is a target for inhibition of urease-related pathology. When our ion mobility-mass spectrometry datasets are combined with previously published chemical crosslinking and x-ray scattering data, a discrete population of conformations for the urease pre-activation complex emerges which compares favorably to previous models generated using computational techniques. In Chapter 6, I highlight more applications of ion mobility-mass spectrometry to engineered and naturally occurring protein complexes. These applications highlight the power of ion mobility mass-mass spectrometry datasets for rapid analysis of protein oligomerization state and structure, providing a basis for further integration of the technology into pharmaceutical and structural biology workflows.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138785/1/joeesch_1.pd

Hydrogen Exchange Mass Spectrometry for Studying Protein-Ligand Interactions

Hydrogen deuterium exchange (HDX) coupled with mass spectrometry is widely used for probing protein structure and dynamics. Protein-ligand interactions usually induce a reduction in the measured HDX rates an effect that may be ascribed to stabilization of the protein structure. This work aims to improve the general understanding of the changes in HDX patterns associated with ligand binding. We initially applied HDX for studying differences between oxy-hemoglobin (Oxy-Hb) and aquomet-hemoglobin (Chapter 2). The results show that the α and β subunits respond differently to the oxy to aquomet transition with the heme binding pocket being destabilized in both cases. The results suggest that enhanced structural dynamics in the heme binding pocket may have adverse effects on heme-protein interactions. Chapter 3 focuses on the different scenarios that can be encountered in an HDX experiment upon ligand binding. Myoglobin and hemoglobin were used as model systems, focusing on the oxy and deoxy states of both proteins. Our results demonstrate that ligand binding can be stabilizing or destabilizing, leading to decreased or increased HDX rates respectively. In Chapters 4 HDX was used to probe the changes in structural dynamics of caseinolytic protease P (ClpP), an antibiotic drug target, after binding ADEP antibiotics. The mechanism of ADEP binding and the N-terminal structure of ClpP is not well understood with conflicting x-ray structures reported in literature. Our findings demonstrate that the N-terminus of ClpP remains quite unstructured after ADEP binding, while belt region undergoes tightening. Pin 1, a peptidyl prolyl isomerase, binding to a cyclic peptide inhibitor was studied in Chapter 5. Characterization of Pin1-CRYPEVEIC interactions by other techniques has been iv difficult. This study demonstrates that binding of the inhibitor triggers an overall stabilization of Pin 1. We identify a loop that interacts with basic sites of the ligand and that becomes destabilized upon ligand binding. This destabilization is ascribed to steric clashes between the peptide inhibitor and the protei

Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115–125 within the yeast Top1p N-terminal domain are required for the complementation of the top1Δ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity

Hydrogels for Regenerative Medicine

Regenerative medicine requires materials that are biodegradable, biocompatible, structurally and chemically stable, and that can mimic the properties of the native extracellular matrix (ECM). Hydrogels are hydrophilic three-dimensional networks that have long received attention in the field of regenerative medicine due to their unique properties. Hydrogels have a potential to be the future of regenerative medicine due to their desirable mechanical and chemical properties, ease of their synthesis, and their multiple applicability as drug delivery vehicles, scaffolds, and constructs for cell culture. In this chapter, we have described hydrogels in terms of their cross-linking and then discussed the most recent developments in the use of hydrogels for peripheral nerve regeneration, tooth regeneration, and 3D bioprinting

Structural characterization of protein-DNA response element ternary complex

openI fattori di trascrizione sono elementi fondamentali alla base di diversi processi biologici, tra cui la regolazione del ciclo cellulare e del differenziamento cellulare durante lo sviluppo, il controllo della risposta immunitaria e il mantenimento dell’omeostasi cellulare. La loro capacità di regolare la trascrizione di specifici geni permette alla cellula di rispondere a diversi stimoli esterni. Sono elementi dinamici che intervengono alla fine della cascata di trasduzione quando, attivati da diverse reazioni chimiche, legano il DNA e ne permettono la trascrizione. L‘interazione con il DNA avviene per la maggior parte dei casi in collaborazione con altri fattori di trascrizione o numerosi cofattori. Alterazioni di questo meccanismo portano all‘insorgenza di molteplici patologie tra cui il cancro. Per questo motivo, l’intricata rete in cui partecipano i fattori di trascrizione con i cofattori e le interazioni che creano è un campo della ricerca attivo. In questa tesi, due fattori di trascrizione, in particolare TEAD1 e FOXO4 sono stati analizzati per ottenere informazioni riguardo cambi conformazionali che avvengono nell’interazione con il DNA e nell’interazione proteina-proteina in assenza o in presenza del DNA. Basandosi su precedenti evidenze di una possibile interazione tra TEAD1 e FOXO4, è stato deciso di svolgere l’esperimento coniugando una reazione di cross-linking con l’analisi LC-MS. Le due proteine sono state analizzate in condizioni differenti utilizzando DSA cross-linker marcato con isotopi nella sua versione leggera e pesante. Conoscendo la posizione degli aminoacidi coinvolti nel legame con il cross-linking agent e sapendo che il cross-linking agent ha una lunghezza definita che determina la distanza tra i gruppi funzionali che lega, è stato possibile ottenere informazioni su come TEAD e FOX04 riarrangiano la loro conformazione Oltretutto, DSA cross-linker ha permesso, essendo marcato con isotopi, la quantificazione dei cambiamenti conformazionali avvenuti nelle proteine nelle condizioni studiate e le dinamiche di interazione delle proteine. Sono stati caratterizzati i cambi conformazionali delle proteine nella sola presenza del DNA. Nel caso di TEAD1, 7 cross-links sono stati individuati e tra questi, tre in particolare indicano che TEAD cambia conformazione nell‘interazione con il DNA. Al contrario, per FOXO4 i tre cross-links individuati rimangono quantitativamente invariati anche quando il fattore di trascrizione lega il DNA Inoltre, quando TEAD1 e FOXO4 sono in soluzione insieme, nell’aggiunta del DNA `e stato notata un’interazione tra le due proteine per formare un complesso ternario TEAD1-FOXO4-DNA. La presenza significativa del cross-link ha confermato quest’interazione.Transcription factors are key elements of several biological processes such as control of cell cycle progression, differentiation of cells during development, immune response, or maintenance of intracellular metabolic balance. Their ability to regulate the transcription of specific sets of genes makes the cells responsive to diverse stimuli. They are dynamic elements participating in the last step of a signalling cascade with the ability to bind the DNA cooperating with other transcription factors and with numerous cofactors. They are elements of interest since dysregulation of the transcription leads to many diseases including cancer. For this reason, the complicated network of transcription factors and cofactors and their interactions is an active research field. In this work, two transcription factors TEAD and FOXO4 were analysed to retrieve some information about their structural change in conformation when they are interacting with the DNA response element and when they are in a mixture together either with or without the DNA. Based on previous evidence of a possible interaction between TEAD and FOXO4, the experiment was set on the use of isotopic cross-linkers conjugated with LC-MS analysis. The two proteins were studied in different conditions in the presence of the isotopically labelled DSA cross-linking agents. The positions of the cross-linked amino acids combined with the known cross-linking agent length that imposes a distance constraint between functional groups within a protein or a complex gave information on the 3D structure of TEAD, FOXO4 and their complex. Moreover, the isotopically labelled DSA cross-linker allowed the quantification of conformational changes of proteins under different conditions and the protein interaction dynamics. It was identified how the proteins change conformation when they are in the only presence of DNA. In the case of TEAD, 7 cross-links were depicted and among them, 3 highlighted a change in the structure of the protein in the presence of the DNA. On the contrary, for FOXO4 3 cross-links were identified which are slightly changing when the transcription factor interacts with the DNA. Furthermore, when TEAD and FOXO4 are mixed in the presence of the DNA, it was noticed that they interact and change conformation to adjust themselves in the formation of a ternary complex TEAD-FOXO4-DNA and a significant presence of a cross-link confirmed an interaction of TEAD and FOXO4 to form the complex with the DNA