About

Stephen D. Fried is a native of Kansas City who received two S.B. degrees in chemistry and physics from MIT in 2009. He completed his doctoral training at Stanford University under the mentorship of Professor S. G. Boxer in 2014, where his graduate research focused on understanding the physical principles underpinning enzymes' catalytic power. From 2014 to 2018, Stephen was a Junior Research Fellow at King’s College and conducted research at the MRC Laboratory of Molecular Biology in Cambridge, United Kingdom. In 2018, he joined the Johns Hopkins University Department of Chemistry as an assistant professor. He currently holds appointments with the T. C. Jenkins Department of Biophysics and the Department of Biology, and is affiliated with the CMDB, PMB, and CBI training programs. Stephen has received numerous awards including the NIH Director’s New Innovator award, an NSF CAREER award, a Cottrell Scholar award, a Camille Dreyfus Teacher-Scholar award, and a Sloan Fellowship. Outside of his professional work, he enjoys cooking, lifting, and traveling.

Research topics

• Chemistry
• Biology
• Biophysics
• Genetics
• Cell biology
• Computational biology
• Biochemistry
• Organic chemistry
• Stereochemistry
• Quantum mechanics
• Physics
• Medicine
• Chemical physics
• Atomic physics
• Computational chemistry
• Evolutionary biology

Selected publications

• Phase separation of PGL-3 driven by structured domains that oligomerize and interact with RGG motifs

  EMBO Reports · 2026-03-20

  articleOpen access

  Phase separation (PS) of biomolecular condensates is often assumed to be driven by interactions involving nucleic acids and intrinsically disordered regions (IDRs) of proteins. PGL-3 is a component of P granules, biomolecular condensates in C. elegans, that contains two structured domains (D1-D2), an internal IDR, and a C-terminal IDR rich in RGG motifs. Theoretical and in vitro studies implicated the internal IDR and RGG motifs in driving PGL-3 PS via self-interactions and binding to RNA. Studies in cells, however, implicated the D1 and D2 domains. Here, we investigate the molecular basis of PGL-3 PS in vitro using microscopy, crosslinking mass spectrometry, and biophysical measurements. We find that D1-D2 forms oligomers and is necessary and sufficient for PS. The terminal RGG region interacts with D1-D2 in a manner that enhances PS even in the absence of RNA. In contrast, the internal IDR is neither necessary nor sufficient for PS. These findings support an alternative model for PGL-3 PS that does not require RNA and is driven by oligomerization of structured domains that interact with RGG repeats.

  PublisherOA PDFDOI

• BPS2026 – In vitro reconstitution of the C. elegans synaptonemal complex

  Biophysical Journal · 2026-02-01

  article
  PublisherDOI

• Limited Proteolysis Mass Spectrometry to Identify Protein Structural Differences in Brain Tissue

  BIO-PROTOCOL · 2026-01-01

  articleOpen accessSenior author

  Structural proteomics methods allow for the proteome-wide interrogation of protein structural differences between two different conditions. Limited proteolysis mass spectrometry (LiP-MS), as originally implemented by the Picotti lab, utilizes a promiscuous protease to cleave at solvent-exposed regions of a protein to encode structural information, which is then read out with mass spectrometry proteomics. Here, we present a protocol that details experimental steps and data analysis for a LiP-MS workflow. First, tissue is homogenized under native conditions and then subjected to limited proteolysis using proteinase K (PK). The samples are prepared for mass spectrometry, and data are acquired using either data-dependent acquisition (DDA) or data-independent acquisition (DIA). Raw data is processed using FragPipe, and raw ion abundances are processed in FragPipe Limited-Proteolysis Processor (FLiPPR). Proteins with structural changes between the two conditions are identified in a proteome-wide manner. Key features • Protocol describes how to perform limited proteolysis mass spectrometry to identify proteins in brain tissue with structural changes proteome-wide between two experimental conditions. • Includes context for how to ensure results are reliable, using permutation analyses. • Utilizes tools (FragPipe and FLiPPR) that are free and open source. • Sample preparation can be performed in two days, not including mass spec acquisition and data analysis.

  PublisherDOI

• Reconstructing ancient protein alphabets: what can we truly infer?

  Trends in Genetics · 2026-03-01

  articleOpen access1st authorCorresponding

  The canonical amino acid alphabet has remained remarkably stable since life's early stages, yet the factors that shaped its emergence remain debated. Early views emphasized prebiotic availability and the expansion of metabolic pathways, but recent advances (particularly from protein biophysics and deep phylogenetics) have brought new perspectives to this question. Together, these views agree that the canonical alphabet emerged from a chemically restricted repertoire and was gradually tightened by metabolic innovation and selection for foldable, functional proteins. However, these approaches can yield inconsistent trajectories, underscoring how much remains unresolved. Here, we compare insights from four lines of evidence, highlight their limitations, and argue that our chronology of amino acid recruitment should be based on where the approaches converge.

  PublisherDOI

• Phosphoglycerate Kinase Can Adopt Topologically Misfolded Forms That Are More Stable Than Its Native State

  Journal of the American Chemical Society · 2026-01-06 · 1 citations

  articleSenior authorCorresponding

  The native states of globular proteins are typically viewed as being the most stable conformations on their respective proteins’ soluble free energy landscapes. This view, known as the thermodynamic hypothesis, explains why many proteins can reversibly refold after being denatured. Here we report an intriguing counterexample to this paradigm. When E. coli phosphoglycerate kinase (PGK) is allowed to refold upon dilution from denaturant, instead of returning to its native state, it populates an unusual misfolded form that is monomeric and native-like, but which is even more kinetically stable than its native form, as based on its resistance to thermal and detergent-induced denaturation. Moreover, this misfolded form cannot self-correct, even for days. We show that the key structural feature of this misfolded form of PGK is topological in nature by demonstrating that kinetically stable misfolded forms do not form any longer if PGK is circularized, which prevents its termini from threading through other portions of the protein. Our findings demonstrate that a misfolded protein need not aggregate or form an amyloid to become stabilized with respect to the native state, and call attention to topologically misfolded proteins as a potential Achilles heel to the cellular proteostasis network.

  PublisherDOI

• Chaperone Dependency during Primary Protein Biogenesis Does Not Correlate with Chaperone Dependency during <i>in vitro</i> Refolding

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-19 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract Many proteins require molecular chaperones to fold into their functional native forms. Previously we used limited proteolysis mass-spectrometry (LiP-MS) to find that ca. 40% of the E. coli proteome do not efficiently refold spontaneously following dilution from denaturation, a frequency that drops to ca. 15% once molecular chaperones like DnaK or GroEL are provided. However, the roles of chaperones during primary biogenesis in vivo can differ from the functions they play during in vitro refolding experiments. Here, we used LiP-MS to probe structural changes incurred by the E. coli proteome when two key chaperones, trigger factor and DnaKJ, are deleted. While knocking out DnaKJ induces pervasive structural perturbations across the soluble E. coli proteome, trigger factor deletion only impacts a small number of proteins’ structures. Overall, proteins which cannot spontaneously refold (or require chaperones to refold in vitro ) are not more likely to be dependent on chaperones to fold in vivo . For instance, the glycolytic enzyme, phosphoglycerate kinase (PGK), cannot refold to its native form in vitro following denaturation (even with chaperones), but by LiP-MS we find that its structure is unperturbed upon DnaKJ or Tig deletion, which is further supported with biochemical and biophysical assays. Thus, PGK folds to its native structure most efficiently during co-translational folding and does so without chaperone assistance. This behaviour is generally found among chaperone-nonrefolders (proteins that cannot refold even with chaperone assistance), strengthening the view that chaperone-nonrefolders are obligate co-translational folders. Hence, for some E. coli proteins, the vectorial nature of co-translational folding is the most important “chaperone.” Highlights - LiP-MS is used to identify which proteins in E. coli are structurally perturbed when DnaKJ or trigger factor is deleted - Very few proteins require trigger factor to assume their native structures - The proteome’s dependence on DnaKJ is increased at lower growth temperature - The enzyme PGK does not need chaperones to fold, but it cannot refold from denaturant, even with chaperone assistance - For some E. coli proteins (such as PGK) co-translational folding during primary biogenesis is the most important “chaperone”

  PublisherDOI

• A Roadmap for Improving Data Reliability and Sharing in Crosslinking Mass Spectrometry

  ArXiv.org · 2025-04-09

  preprintOpen access

  Crosslinking Mass Spectrometry (MS) can uncover protein-protein interactions and provide structural information on proteins in their native cellular environments. Despite its promise, the field remains hampered by inconsistent data formats, variable approaches to error control, and insufficient interoperability with global data repositories. Recent advances, especially in false discovery rate (FDR) models and pipeline benchmarking, show that Crosslinking MS data can reach a reliability that matches the demand of integrative structural biology. To drive meaningful progress, however, the community must agree on error estimation, open data formats, and streamlined repository submissions. This perspective highlights these challenges, clarifies remaining barriers, and frames practical next steps. Successful field harmonisation will enhance the acceptance of Crosslinking MS in the broader biological community and is critical for the dependability of the data, no matter where it is produced.

  PublisherOA PDFDOI

• Investigation of the global translational response to oxidative stress in the model archaeon <i>Haloferax volcanii</i> reveals untranslated small RNAs with ribosome occupancy

  mSphere · 2025-09-08

  articleOpen access

  ABSTRACT Oxidative stress induces a wide range of cellular damage, often causing disease and cell death. While many organisms are susceptible to the effects of oxidative stress, haloarchaea have adapted to be highly resistant. Several aspects of the haloarchaeal oxidative stress response have been characterized; however, little is known about the impacts of oxidative stress at the translation level. Using the model archaeon Haloferax volcanii , we performed RNA-seq and ribosome profiling (Ribo-seq) to characterize the global translation landscape during oxidative stress. We identified 281 genes with differential translation efficiency (TE). Downregulated genes were enriched in ribosomal and translation proteins, in addition to peroxidases and genes involved in the TCA cycle. We also identified 42 small noncoding RNAs (sRNAs) with ribosome occupancy. Size distributions of ribosome footprints revealed distinct patterns for coding and noncoding genes, with 12 sRNAs matching the pattern of coding genes, and mass spectrometry confirming the presence of seven small proteins encoded by these sRNAs. However, the majority of sRNAs with ribosome occupancy had no evidence of coding potential. Of these ribosome-associated sRNAs, 12 had differential ribosome occupancy or TE during oxidative stress, suggesting that they may play a regulatory role during the oxidative stress response. Our findings on ribosomal regulation during oxidative stress, coupled with potential roles for ribosome-associated noncoding sRNAs and sRNA-derived small proteins in H. volcanii , revealed additional regulatory layers and underscored the multifaceted architecture of stress-responsive regulatory networks. IMPORTANCE Archaea are found in diverse environments, including as members of the human microbiome, and are known to play essential ecological roles in major geochemical cycles. The study of archaeal biology has expanded our understanding of the evolution of eukaryotes, uncovered novel biological systems, and revealed new opportunities for applications in biotechnology and bioremediation. Many archaeal systems, however, remain poorly characterized. Using Haloferax volcanii as a model, we investigated the global translation landscape during oxidative stress. Our findings expand current knowledge of translational regulation in archaea and further illustrate the complexity of stress-responsive gene regulation.

  PublisherDOI

• Investigation of the global translational response to oxidative stress in the model archaeon <i>Haloferax volcanii</i> reveals untranslated small RNAs with ribosome occupancy

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-08

  preprintOpen access

  Abstract Oxidative stress induces a wide range of cellular damage, often causing disease and cell death. While many organisms are susceptible to the effects of oxidative stress, haloarchaea have adapted to be highly resistant. Several aspects of the haloarchaeal oxidative stress response have been characterized, however little is known about the impacts of oxidative stress at the translation level. Using the model archaeon Haloferax volcanii , we performed RNA-seq and ribosome profiling (Ribo-seq) to characterize the global translation landscape during oxidative stress. We identified 281 genes with differential translation efficiency (TE). Downregulated genes were enriched in ribosomal and translation proteins, in addition to peroxidases and genes involved in the TCA cycle. We also identified 42 small noncoding RNAs (sRNAs) with ribosome occupancy. Size distributions of ribosome footprints revealed distinct patterns for coding and noncoding genes, with 12 sRNAs matching the pattern of coding genes, and mass spectrometry confirming the presence of seven small proteins encoded in these sRNAs. However, the majority of sRNAs with ribosome occupancy had no evidence of coding potential. Of these ribosome-associated sRNAs, 12 had differential ribosome occupancy or TE during oxidative stress, suggesting that they may play a regulatory role during the oxidative stress response. Our findings on ribosomal regulation during oxidative stress, coupled with potential roles for ribosome-associated noncoding sRNAs and sRNA-derived small proteins in H. volcanii , revealed additional regulatory layers and underscore the multifaceted architecture of stress-responsive regulatory networks. Importance Archaea are found in diverse environments, including as members of the human microbiome, and are known to play essential ecological roles in major geochemical cycles. The study of archaeal biology has expanded our understanding of the evolution of eukaryotes, uncovered novel biological systems, and revealed new opportunities for applications in biotechnology and bioremediation. Many archaeal systems, however, remain poorly characterized. Using Haloferax volcanii as a model, we investigated the global translation landscape during oxidative stress. Our findings expand current knowledge of translational regulation in archaea and further illustrate the complexity of stress-responsive gene regulation.

  PublisherOA PDFDOI

• Chaperone dependency during biogenesis does not correlate with chaperone dependency during refolding

  Molecular Systems Biology · 2025-10-28 · 3 citations

  articleOpen accessSenior author

  Many proteins require molecular chaperones to fold into their functional native forms. However, the roles of chaperones during primary biogenesis in vivo can differ from the functions they play during in vitro refolding experiments. Here, we use limited proteolysis mass spectrometry (LiP-MS) to probe structural changes incurred by the E. coli proteome when two key chaperones, trigger factor and DnaKJ, are deleted. While knocking out DnaKJ induces pervasive structural perturbations across the soluble E. coli proteome, trigger factor deletion only impacts a small number of proteins' structures. Overall, proteins which cannot spontaneously refold (or require chaperones to refold in vitro) are not more likely to be dependent on chaperones to fold in vivo. We find that chaperone-nonrefolders (proteins that cannot refold even with chaperone assistance) do not generally require chaperones to fold in vivo, strengthening the view that chaperone-nonrefolders are obligate co-translational folders. Hence, for some E. coli proteins, the vectorial nature of co-translational folding is the most important "chaperone".

  PublisherOA PDFDOI

Recent grants

• Watching Proteins Fold (or Misfold) in vivo with Mass Spectrometry

  NIH · $2.3M · 2020–2025

• CAREER: Leaving the Fold: Leveraging Mass Spectrometry Proteomics to Shift the Paradigm on Protein Folding

  NSF · $852k · 2021–2025

Frequent coauthors

• Steven G. Boxer

  Stanford University

  24 shared

• Jason W. Chin

  Medical Research Council

  19 shared

• Philip To

  Johns Hopkins University

  14 shared

• Edgar Manriquez‐Sandoval

  Johns Hopkins University

  13 shared

• Chayasith Uttamapinant

  Vidyasirimedhi Institute of Science and Technology

  12 shared

• Wolfgang H. Schmied

  MRC Laboratory of Molecular Biology

  12 shared

• Anneliese M. Faustino

  Johns Hopkins University

  11 shared

• Haley E. Tarbox

  10 shared

Labs

• The Fried LabPI

Education

• Ph.D., Chemistry

  Stanford University

  2014

• S.B., Chemistry

  Massachusetts Institute of Technology

  2009

Awards & honors

• HFSP Young Investigator Award
• NIH Director’s New Innovator Award