About

The Pielak Research Group at the University of North Carolina at Chapel Hill focuses on using the formalisms of equilibrium thermodynamics and the tools of molecular biology and biophysics to understand the structure, stability, and function of globular, intrinsically-disordered proteins and protein-protein interactions under physiologically-relevant conditions. This research aims to elucidate fundamental aspects of protein behavior in environments that closely mimic those found in living organisms. The group is led by Professor Gary Pielak, who directs investigations into the biophysical properties of proteins, contributing to a deeper understanding of their roles in cellular processes.

Research topics

• Biochemistry
• Biophysics
• Biology
• Chemistry
• Physics
• Organic chemistry
• Nuclear magnetic resonance
• Thermodynamics
• Cell biology
• Materials science
• Chemical physics
• Stereochemistry

Selected publications

• BPS2026 – Thermal stability of AI-designed protein crowders

  Biophysical Journal · 2026-02-01

  articleSenior author
  PublisherDOI

• Sugar–protein interactions control protein‐complex stability in crowded Ficoll and dextran solutions

  UNC Libraries · 2026-03-14

  articleOpen access

  Traditional views of crowding emphasize excluded volume-stabilization of compact states-because they occupy less space, but the effects of high cosolute concentrations comprise several conjoined processes. We systematically explored the effects of dextrans, Ficolls, and their monomers, glucose and sucrose on the equilibrium thermodynamics of two protein complexes, both variants of the B1 domain of streptococcal protein G, a well-studied, small (6.2 kDa) globular protein. One is a simple side-by-side dimer of folded monomers. The other is a domain swap dimer, where, in buffer, the folded dimer dissociates to partially folded monomers. Dissociation was monitored as a function of cosolute molecular weight, cosolute concentration, and temperature using ¹⁹F NMR. Model fitting shows that, in contrast to sugar monomers whose interaction distance is dictated by their molecular volume, interaction distances of sugar polymers are dictated by their polymer structure and concentration. At high concentrations the effective length scale is the mesh size, emphasizing the decreasing effectiveness of large synthetic polymers. The model further dissects the measured parameters into fundamental processes: excluded volume, chemical interactions, and non-ideal mixing. For glucose and sucrose, dimer-stabilizing excluded volume contributions are nearly completely offset by destabilizing attractive chemical interactions with the monomers. As polymer molecular weight increases, the destabilizing effect of non-ideal mixing completely offsets the stabilizing effects of excluded volume. The results show that the crowding mechanism for polymers is distinct from those of their monomers and reflect their structural properties.

  PublisherDOI

• Trifluoroethanol and the behavior of a tardigrade desiccation‐tolerance protein

  UNC Libraries · 2026-04-14

  articleOpen access1st authorCorresponding

  The cosolvent 2,2,2-trifluoroethanol (TFE) is often used to mimic protein desiccation. We assessed the effects of TFE on cytosolic abundant heat soluble protein D (CAHS D) from tardigrades. CAHS D is a member of a unique protein class that is necessary and sufficient for tardigrades to survive desiccation. We find that the response of CAHS D to TFE depends on the concentration of both species. Dilute CAHS D remains soluble and, like most proteins exposed to TFE, gains α-helix. More concentrated solutions of CAHS D in TFE accumulate β-sheet, driving both gel formation and aggregation. At even higher TFE and CAHS D concentrations, samples phase separate without aggregation or increases in helix. Our observations show the importance of considering protein concentration when using TFE.

  PublisherDOI

• Crowding and Confinement Can Oppositely Affect Protein Stability

  UNC Libraries · 2026-04-08

  articleOpen accessSenior author

  Proteins encounter crowded and confined macromolecular milieus in living cells. Simple theory predicts that both environments entropically stabilize proteins if only hard-core repulsive interactions are considered. Recent studies show that chemical interactions between the surroundings and the test protein also play key roles such that the overall effect of crowding or confinement is a balance of hard-core repulsions and chemical interactions. There are, however, few quantitative studies. Here, we quantify the effects of crowding and confinement on the equilibrium unfolding thermodynamics of a model globular protein, KH1. The results do not agree with predictions from simple theory. KH1 is stabilized by synthetic-polymer crowding agents but destabilized by confinement in reverse micelles. KH1 is more entropically stabilized and enthalpically destabilized in concentrated solutions of the monomers than it is in solutions of the corresponding polymers. When KH1 is confined in reverse micelles, the temperature of maximum stability decreases, the melting temperature decreases, and the protein is entropically destabilized and enthalpically stabilized. Our results show the importance of chemical interactions to protein folding thermodynamics and imply that cells utilize chemical interactions to tune protein stability.

  PublisherDOI

• Sugar–protein interactions control protein‐complex stability in crowded Ficoll and dextran solutions

  Protein Science · 2025-12-22 · 1 citations

  articleOpen accessSenior authorCorresponding

  Abstract Traditional views of crowding emphasize excluded volume—stabilization of compact states—because they occupy less space, but the effects of high cosolute concentrations comprise several conjoined processes. We systematically explored the effects of dextrans, Ficolls, and their monomers, glucose and sucrose on the equilibrium thermodynamics of two protein complexes, both variants of the B1 domain of streptococcal protein G, a well‐studied, small (6.2 kDa) globular protein. One is a simple side‐by‐side dimer of folded monomers. The other is a domain swap dimer, where, in buffer, the folded dimer dissociates to partially folded monomers. Dissociation was monitored as a function of cosolute molecular weight, cosolute concentration, and temperature using 19 F NMR. Model fitting shows that, in contrast to sugar monomers whose interaction distance is dictated by their molecular volume, interaction distances of sugar polymers are dictated by their polymer structure and concentration. At high concentrations the effective length scale is the mesh size, emphasizing the decreasing effectiveness of large synthetic polymers. The model further dissects the measured parameters into fundamental processes: excluded volume, chemical interactions, and non‐ideal mixing. For glucose and sucrose, dimer‐stabilizing excluded volume contributions are nearly completely offset by destabilizing attractive chemical interactions with the monomers. As polymer molecular weight increases, the destabilizing effect of non‐ideal mixing completely offsets the stabilizing effects of excluded volume. The results show that the crowding mechanism for polymers is distinct from those of their monomers and reflect their structural properties.

  PublisherOA PDFDOI

• Disordered proteins mitigate the temperature dependence of site-specific binding free energies

  UNC Libraries · 2025-07-26

  articleOpen accessSenior author
  PublisherDOI

• Effects of Lyophilization, Vacuum Drying, and Microglassification on Two Model Proteins Assessed at the Residue Level Using Liquid Observed Vapor Exchange Nuclear Magnetic Resonance Spectroscopy (LOVE NMR)

  Molecular Pharmaceutics · 2025-07-10 · 2 citations

  articleSenior authorCorresponding

  We explore the effects of drying methods on residue-level protein structure using Liquid-Observed Vapor Exchange Nuclear Magnetic Resonance spectroscopy (LOVE NMR) data from two proteins, the B1 domain of streptococcal protein G and the enzyme adenylate kinase (AdK) from Escherichia coli. The data show that both vacuum drying and microglassification are more protective than lyophilization. Assessing the effects on AdK activity leads to the same conclusion. Another important conclusion comes from comparing solution stability to dry-state protection. Namely, regions exposed only upon complete unfolding in solution are those that are most protected in the dry state, an observation that could be made because of the residue-level resolution of LOVE NMR. The results will help guide the discovery and optimization of new excipients for solid formulations.

  PublisherDOI

• Crowding beyond excluded volume: A tale of two dimers

  Protein Science · 2025-03-17 · 9 citations

  articleOpen accessCorresponding

  Abstract Protein–protein interactions are modulated by their environment. High macromolecular solute concentrations crowd proteins and shift equilibria between protein monomers and their assemblies. We aim to understand the mechanism of crowding by elucidating the molecular‐level interactions that determine dimer stability. Using 19 F‐NMR spectroscopy, we studied the effects of various polyethylene glycols (PEGs) on the equilibrium thermodynamics of two protein complexes: a side‐by‐side and a domain‐swap dimer. Analysis using our mean‐field crowding model shows that, contrary to classic crowding theories, PEGs destabilize both dimers through enthalpic interactions between PEG and the monomers. The enthalpic destabilization becomes more dominant with increasing PEG concentration because the reduction in PEG mesh size with concentration diminishes the stabilizing effect of excluded volume interactions. Additionally, the partially folded domain‐swap monomers fold in the presence of PEG, contributing to dimer stabilization at low PEG concentrations. Our results reveal that polymers crowd protein complexes through multiple conjoined mechanisms, impacting both their stability and oligomeric state.

  PublisherOA PDFDOI

• Crowding‐induced stabilization and destabilization in a single protein

  Protein Science · 2025-04-22 · 5 citations

  articleOpen accessSenior authorCorresponding

  Abstract The protein concentration in cells can reach 300 g/L. These crowded conditions affect protein stability. Classic crowding theories predict entropically driven stabilization, which occurs via steric repulsion, but growing evidence shows a role for non‐covalent chemical interactions. To aid our understanding of physiologically relevant crowding, we used NMR‐detected 1 H‐ 2 H exchange to examine a simple, semi‐reductionist system: protein self‐crowding at the residue level using the widely studied model globular protein, GB1 (the B1 domain streptococcal protein G) at concentrations up to its solubility limit, 100 g/L. The surprising result is that self‐crowding stabilizes some residues but destabilizes others, contradicting predictions. Two other observations are also contradictory. First, temperature‐dependence data show that stabilization can arise enthalpically, not just entropically. Second, concentration‐dependence data show destabilization often increases with increasing concentration. These results show a key role for chemical interactions. More specifically, self‐crowding increases the free energy required to expose those residues that are only exposed upon complete unfolding, and stabilization of these globally unfolding residues increases with GB1 concentration, a result we attribute to repulsive chemical interactions between GB1 molecules. On the other hand, residues exposed upon local unfolding tend to be destabilized, with destabilization increasing with concentration, a result we attribute to attractive chemical interactions between GB1 molecules.

  PublisherOA PDFDOI

• Understanding dry proteins and their protection with solid‐state hydrogen–deuterium exchange

  Protein Science · 2025-02-25

  reviewOpen accessSenior author

  Protein-based drugs are among our most powerful therapeutics, but their manufacture, storage, and distribution are hindered by solution instability and the expense of the necessary refrigeration. Formulating proteins as dry products, which is an almost entirely empirical endeavor, can ameliorate the problem, but recovery of an acceptable product upon resuspension is not always possible. Additional knowledge about dry protein structure and protection is necessary to make dry formulation both more rational and effective. While most biophysical and biochemical techniques necessitate solvated protein, solid-state hydrogen-deuterium exchange enables the study of dry proteins. Fourier-transform infrared spectroscopy, mass spectrometry, and liquid-observed vapor exchange nuclear magnetic resonance have all been used to measure isotopic exchange. These methods report on secondary structure, peptide, and residue level exposure, respectively. Recent studies using solid-state hydrogen-deuterium exchange provide insight into the mechanisms of dry protein protection and uncover stabilizing and destabilizing interactions, bringing us closer to rational formulation of these lifesaving products.

  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM042501

  NIH · $722k · 2000

• Encapsulation and Protein Stability

  NSF · $418k · 2016–2019

• Protein stabilizers from tardigrades

  NIH · $1.4M · 2018–2022

• NIH Director's Pioneer Award

  NIH · $3.7M · 2006–2013

• NIH Grant R21ES010774

  NIH · $245k · 2003

Frequent coauthors

• Conggang Li

  Wuhan Institute of Physics and Mathematics

  118 shared

• Maili Liu

  University of Chinese Academy of Sciences

  66 shared

• Gregory B. Young

  University of North Carolina at Chapel Hill

  45 shared

• Wenwen Zheng

  Shanghai Institute of Ceramics

  33 shared

• Samantha Piszkiewicz

  Pivot Bio (United States)

  31 shared

• Austin E. Smith

  Cardiff and Vale University Health Board

  30 shared

• Yaqiang Wang

  University of California, Los Angeles

  29 shared

• Ling Jiang

  Huazhong University of Science and Technology

  28 shared

Labs

• Pielak Research GroupPI

Education

• Ph.D., Biochemistry

  University of California, San Francisco

  1990

• B.S., Chemistry

  University of California, Santa Barbara

  1985

Awards & honors

• Biophysical Society Fellow, 2024
• Excellence in Doctoral Mentoring Award, UNC, 2023
• Johnston Teaching Excellence Award, UNC, 2023
• Award for Excellence in Basic Science Mentoring, UNC Office…
• Glen H. Elder, Jr. Distinguished Term Professor of Research…