About

Vincent Hilser is a professor and chair of the Department of Biology at Johns Hopkins University. He received his PhD from Johns Hopkins University and his research focuses on conformational fluctuations and intrinsic disorder in allosteric signaling, disease, and evolution. His lab is interested in elucidating the structural and energetic basis of fluctuations, as well as their functional consequences, and applying this information to the development of protein design and optimization strategies and novel fold classification and genomic approaches. His research involves developing and testing structure-based models of conformational fluctuations that can capture a broad spectrum of biophysical and functional phenomena within a unified framework. The goal is to quantitatively link fluctuations to folding and stability, investigating the complex interplay between ligand binding, global structural transitions, and fluctuations. To challenge and refine these models, his lab employs various experimental systems, including titration and scanning calorimetry, NMR spectroscopy, X-ray crystallography, CD, and fluorescence spectroscopy, providing both global and site-resolved characterizations of proteins.

Research topics

• Biology
• Biophysics
• Chemistry
• Genetics
• Computer Science
• Biochemistry
• Cell biology
• Paleontology
• Computational biology
• Evolutionary biology
• Cancer research

Selected publications

• Multivalent weak contacts shape chaperone-nascent protein interactions

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-03-10

  articleOpen access

  Molecular chaperones interact with non-native proteins, playing crucial roles in preventing misfolding and enable efficient folding in the cellular environment. Trigger factor is a bacterial chaperone that binds to ribosomes, interacting with nascent polypeptides emerging from the ribosome and guiding their early folding steps. In contrast to the central role of the chaperone in promoting folding of newly synthesized proteins, its dynamic interactions with nascent chains emerging from the ribosome remain poorly understood. Here, we use single-molecule fluorescence and optical tweezers approaches to directly observe and characterize trigger factor interactions with a ribosome-bound client protein at increasing chain lengths. We find that trigger factor binding to nascent proteins is best described by a combination of multiple weak, dynamic interactions that are established after the chaperone docks onto the ribosome and evolve during polypeptide elongation. Application of mechanical force perturbs trigger factor binding, supporting a multivalent interaction model. This binding mode may help to stabilize nascent proteins against misfolding while allowing them to dynamically sample conformational space in search of their native structures.

  PublisherOA PDFDOI

• Ensemble molecular mimicry correlates with antibody cross-reactivity in proteome-wide studies

  Frontiers in Immunology · 2026-02-10

  articleOpen accessSenior author

  Energetics of protein-protein binding necessarily include contributions both from conformational equilibria and from interfacial interactions. In the particular case of an antibody binding to a protein epitope, the conformational contribution is typically neglected as the antibody-bound and free forms of the protein are usually highly similar, leading to the reasonable conclusion that binding affinity in most cases can be reconciled in the context of observed interfacial interactions. However, the phenomenon of molecular mimicry has also been widely observed, wherein antibodies raised against one sequence/structure are able to recognize a completely different sequence/structure. This observation suggests that, in some cases, the conformational contribution could play a significant role in facilitating this cross-reactivity. Here, this conjecture is supported, utilizing a recent discovery that permits evaluation of the thermodynamic compatibility of any sequence for the conformational ensemble of any other protein-in effect providing direct access to the conformational contribution to binding. The importance of the contribution could then be assessed on a proteome-wide scale, in the context of the unexpected cross-reactivity observed when the human proteome is challenged with antibodies raised against a set of virus protein antigens. Because the virus protein antigens and the cross-reactive human proteins share substantial similarity when modeled as thermodynamic ensembles, despite the absence of detectable sequence or structural similarity, we hypothesize that these cross-reactive epitopes share a novel kind of immunological molecular mimicry, termed "ensemble molecular mimicry" (EMM). To investigate potential mechanisms, a sequence-based algorithm was developed to probe for the relationship between high scoring sequence segments and cross-reacting epitopes, and it was discovered that 9 of 11 medically relevant cross-reactive epitopes taken from the literature exhibited higher-than-expected local EMM values. Taken together, the results suggest that conformational equilibrium can affect affinity and that it is hypothetically possible for cross-reactive epitopes to share a pairwise thermodynamic signature, even in the absence of sequence or structural similarity.

  PublisherOA PDFDOI

• Impact of local unfolding fluctuations on the evolution of regional sequence preferences in proteins

  Protein Science · 2025-02-19 · 2 citations

  articleOpen accessSenior authorCorresponding

  The number of distinct structural environments in the proteome (as observed in the Protein Data Bank) may belie an organizing framework, whereby evolution conserves the relative stability of different sequence segments, regardless of the specific structural details present in the final fold. If true, the question arises as to whether the energetic consequences of amino acid substitutions, and thus the frequencies of amino acids within each of these so-called thermodynamic environments, could depend less on what local structure that sequence segment may adopt in the final fold, and more on the local stability of that final structure relative to the unfolded state. To address this question, a previously described ensemble-based approach (the COREX algorithm) was used to define proteins in terms of thermodynamic environments, and the naturally occurring frequencies of amino acids within these environments were used to generate statistical energies (a type of knowledge-based potential). By comparing compatibility scores from the statistical energies with energies calculated using the Rosetta all-atom energy function, we assessed the information overlap between the two approaches. Results revealed a substantial correlation between the statistical scores and those obtained using Rosetta, directly demonstrating that a small number of thermodynamic environments are sufficient to capture the perceived multiplicity of different structural environments in proteins. More importantly, the agreement suggests that regional amino acid distributions within each protein in any proteome have been substantially driven by the evolutionary conservation of the regional differences in stabilities within protein families.

  PublisherDOI

• The Ensemble Basis of Allostery and Function: Insights from Models of Local Unfolding

  Journal of Molecular Biology · 2025-06-09 · 5 citations

  reviewOpen accessSenior authorCorresponding
  PublisherOA PDFDOI

• Chaperone saturation mediates translation and protein folding efficiency

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-28

  preprintOpen accessSenior authorCorresponding

  Abstract Whether the emergence of a nascent protein from the ribosome and the formation of structural elements are synchronized has been a longstanding question (Chaney and Clark, 2015; Deane and Saunders, 2011; Levinthal, 1968; Marin, 2008; Sauna and Kimchi-Sarfaty, 2011; Spencer and Barral, 2012; Tsai et al., 2008; Zhang and Ignatova, 2011). Paradoxically, kinetically efficient translation can induce mis-folding and aggregation despite the presence of molecular chaperones (Siller et al., 2010; Spencer et al., 2012), which in Escherichia coli are induced by unfolded protein (Parsell and Sauer, 1989) via σ 32 (Craig and Gross, 1991). The molecular mechanisms mediating translation efficiency and protein folding efficiency remain poorly understood. Using ribosome profiling (Ingolia et al., 2009) and protein quantitation, we show that synonymous changes to Firefly Luciferase ( Luc ) mRNA have a direct effect on its translation efficiency. These changes alone cause up to a 70-fold difference in Luc protein levels. However, increased Luc protein is met with at most a ∼2-fold increase in chaperone levels, revealing that the σ 32 transcriptional response has saturable properties. This response is found to be poised near its midpoint (where it is most sensitive to perturbation) when Luc mRNA has an intermediate translation efficiency. These results suggest not only that chaperone saturation limits the ability of cells to maintain protein folding homeostasis when challenged with highly efficient translation, but that translation efficiency and protein folding efficiency evolved for mutual sensitivity.

  PublisherDOI

• Arrest Peptide Profiling resolves co-translational folding pathways and chaperone interactions in vivo

  Nature Communications · 2025-07-24 · 2 citations

  articleOpen access

  Cytosolic proteins begin to fold co-translationally as soon as they emerge from the ribosome during translation. These early co-translational steps are crucial for overall folding and are guided by an intricate network of interactions with molecular chaperones. Because cellular co-translational folding is challenging to detect, its timing and progression remain largely elusive. To quantitatively define co-translational folding in live cells, we developed a high-throughput method that we term “Arrest Peptide Profiling” (AP Profiling). Combining AP Profiling with single-molecule experiments, we delineate co-translational folding for a set of GTPase domains with similar structures, defining how topology shapes folding pathways. Genetic ablation of nascent chain-binding chaperones results in discrete and localized folding changes, highlighting how functional redundancy among chaperones is achieved by distinct engagement with the nascent protein. Our work provides a window into cellular folding pathways of structurally intricate proteins and paves the way for systematic studies of nascent protein folding at exceptional resolution and throughput. Protein folding is facilitated by cellular machinery, but studying folding in the cellular context is challenging. Here, the authors developed Arrest Peptide Profiling to map co-translational folding and chaperone interactions in living cells.

  PublisherOA PDFDOI

• The Ensemble Basis of Allostery and Function: Insights from Models of Local Unfolding

  SSRN Electronic Journal · 2025-01-01

  preprintOpen accessSenior author
  PublisherDOI

• 934 Altered peptide ligands derived from a public p53 neoantigen confer superior immunogenicity as off-the-shelf vaccine candidates

  Regular and Young Investigator Award Abstracts · 2025-11-01

  articleOpen access
  PublisherOA PDFDOI

• Statistical Thermodynamics of the Protein Ensemble: Mediating Function and Evolution

  Annual Review of Biophysics · 2025-02-10 · 6 citations

  reviewOpen access1st authorCorresponding

  The growing appreciation of native state conformational fluctuations mediating protein function calls for critical reevaluation of protein evolution and adaptation. If proteins are ensembles, does nature select solely for ground state structure, or are conformational equilibria between functional states also conserved? If so, what is the mechanism and how can it be measured? Addressing these fundamental questions, we review our investigation into the role of local unfolding fluctuations in the native state ensembles of proteins. We describe the functional importance of these ubiquitous fluctuations, as revealed through studies of adenylate kinase. We then summarize elucidation of thermodynamic organizing principles, which culminate in a quantitative probe for evolutionary conservation of protein energetics. Finally, we show that these principles are predictive of sequence compatibility for multiple folds, providing a unique thermodynamic perspective on metamorphic proteins. These research areas demonstrate that the locally unfolded ensemble is an emerging, important mechanism of protein evolution.

  PublisherDOI

• CEST NMR characterization of functional domain unfolding in adenylate kinase

  Biophysical Journal · 2024-02-01

  articleSenior author
  PublisherDOI

Recent grants

• Thermodynamics of Denatured State Polyproline II Conformational Bias

  NSF · $687k · 2005–2011

• The Experimental Energy Landscape and Protein Function

  NIH · $7.9M · 2001–2025

• Intrinsic Disorder, Energetic Coupling and Allostery

  NSF · $587k · 2013–2017

• Allostery in an intrinsically disordered transcription factor

  NIH · $1.3M · 2018–2022

• Thermodynamics of Denatured State Polyproline II Conformational Bias

  NSF · $56k · 2010–2011

Frequent coauthors

• James O. Wrabl

  Johns Hopkins University

  38 shared

• Steven T. Whitten

  35 shared

• E. Brad Thompson

  Johns Hopkins University

  16 shared

• Bertrand García‐Moreno E.

  15 shared

• Ernesto Freire

  Johns Hopkins University

  14 shared

• Terrence G. Oas

  14 shared

• Hesam N. Motlagh

  Johns Hopkins University

  11 shared

• Jing Li

  11 shared