A stability study of small cold shock proteins from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima finds that cold unfolding appears as a new but unsuspected factor limiting life at low temperatures.
Abstract
Small cold shock proteins from bacteria are model proteins for many biophysical studies as a result of their small size and compact folding. Here we report a stability study of these proteins from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. When analyzed by differential scanning calorimetry, the three proteins unfolded fully reversibly according to a perfect two-state model, which are strict prerequisites to apply equilibrium thermodynamic equations. The parameters obtained by differential scanning calorimetry allowed to calculate the thermodynamic stability of the three proteins and to draw their bell-shaped stability curves, i.e. the free energy of unfolding as a function of temperature. These curves provided fundamentals of stability for proteins adapted to extreme temperatures. For instance, the maximal stability of the three proteins was reached around room temperature and is not shifted toward high temperature for the hyperthermophilic protein, nor to low temperature for the psychrophilic one. The latter is heat-labile but is also cold-labile. Therefore, cold unfolding appears as a new but unsuspected factor limiting life at low temperatures. The distinct temperature-induced dynamics of these proteins are also discussed.
Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing Gquadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. To understand the structural basis of this catalytic activity, we introduce NMR method to solve the structures, at base-level resolution, of a multiconformer G4 with chaperone activity without chemical shift assignments. We then perform structure-function studies via mutation and chaperone assays to test the G4 properties important for chaperoning protein aggregation and protein folding. Together, our finding uncovers a previously underappreciated role for nucleic acid in proteostasis and offer a new strategy for studying nucleic acid structure-function relationship at residue level.
Understanding how proteins behave at low temperatures remains a central challenge in biophysics, with direct implications for cold denaturation and cryopreservation. While cold denaturation of proteins in the supercooled liquid regime has been studied extensively, the behavior of a protein embedded in a growing ice lattice remains largely inaccessible to experiments. Here we use molecular dynamics simulations that explicitly capture ice Ih formation to characterize the conformational dynamics of yeast frataxin (Yfh1) as its aqueous environment crystallizes. Using four independent ice-seeded replicas and liquid-water controls at three temperatures, we first validate the liquid-solid transition through convergent changes in solvent density, potential energy, and local bond-order parameters (W4, W6). Principal component analysis (PCA), dihedral PCA (dPCA), and free-energy landscapes then reveal that crystallization of the solvent markedly reshapes the accessible conformational space, shifting it from a continuous, highly connected regime in liquid water toward a discretized landscape dominated by confined states. Complementary analyses of solvent-accessible surface area (SASA), radius of gyration, and hydrogen bonding indicate a solvent-driven reorganization of protein-water interactions: although first-shell water remains liquid-like, its surface density increases under freezing, while conformational sampling contracts. Together, these results indicate that protein behavior at low temperatures is governed not by temperature alone but by the structural organization of the surrounding water. By imposing geometrical constraints on the solvent, ice formation restricts conformational sampling while preserving -- and even densifying -- the interfacial hydration layer, highlighting the role of water structure as a determinant of protein stability under freezing conditions.
Yanis R. Espinosa, H. Alvarez, C. M. Carlevaro· 0 citations
Enzyme activity increases with temperature up to a maximum, beyond which it declines, a behaviour traditionally attributed to thermal denaturation. However, some enzymes show activity decline well below the melting temperature. Macromolecular rate theory (MMRT) explains this phenomenon by introducing a negative activation heat capacity , reflecting a transition-state ensemble more conformationally restricted than the ground state. Recently, has been shown to be temperature-dependent and proposed as a general catalytic feature, though its variation within and across homologous families from distinct thermal niches remains unexplored. We characterized the glucokinase activity of three homologous bifunctional ADP-dependent PFK/GK enzymes: MbPFK/GK from the psychrotolerant Methanococcoides burtonii, MmPFK/GK from the mesophilic Methanococcus maripaludis, and ancM, the inferred ancestor of the Methanococcales order, which displays enhanced thermostability. MmPFK/GK and ancM display two regimes, with abrupt changes in kcat vs temperature: zero to moderately negative values at low temperatures, shifting sharply at elevated temperatures to highly negative values (−44 kJ mol−1 K−1 and −36 kJ mol−1 K−1, respectively), exceeding previous reports. Circular dichroism spectroscopy confirms that these extreme values reflect pre-melting conformational changes rather than denaturation. Despite being psychrotolerant, MbPFK/GK displayed the highest thermal stability and a single regime throughout all temperatures (−2.6 kJ mol−1 K−1). Domain-closure dynamics explain thermal adaptation and moderate-temperature values; whereas the basis of the extreme high-temperature values remain unknown. To account for these two regimes, we present a two-pathway model incorporating a conformational equilibrium in which free enzyme and enzyme-substrate complex populate two catalytically competent conformations.
Ignacio Aravena-Valenzuela, P. Maturana, Leslie Hernández-Cabello et al.· bioRxiv· 0 citations
Many bacterial chlorogenic acid esterases (ChlEs) exhibit atypical temperature behavior, featuring activities that barely change with temperature and activity maxima that fall below the thermal denaturation point. This work focuses on a ChlE fromLactobacillus helveticus (Lh–ChlE), which has a flat temperature dependence. First, it was determined that conformational changes during Lh–ChlE turnover are not rate-limiting and that the overall rate depends on the chemical step at all temperatures. Next, Lh–ChlE’s temperature dependence was investigated using a conformational equilibrium model that assumes the existence of a temperature-dependent equilibrium between an active and an inactive conformation and an activation heat capacity model that postulates a difference in heat capacity between the ground and transition states. Although the equilibrium model recapitulates the data well, it yields an unrealistically low inactivation temperature around 280 K. Circular dichroism spectroscopy suggests that Lh–ChlE does not undergo structural changes at that temperature but may undergo small structural transitions at moderately elevated temperature. The activation heat capacity model describes Lh–ChlE behavior well, yielding an activation heat capacity (ΔC p ‡) of approximately −1 kJ mol–1 K–1. Overall, the results suggest that the atypical temperature behavior of Lh–ChlE likely arises from a negative activation heat capacity. This work illustrates that contrasting thermodynamic models for atypical temperature dependence in enzymes can give rise to similar looking fits, even though they have different underlying physical meaning. Our results furthermore encourage additional analysis of the Lh–ChlE transition state structure to better understand the structural features that cause the enzyme’s nonzero activation heat capacity.
Nathaniel B. Carl, Yianni Tsigaris, Dan Ji et al.· Biochemistry· 0 citations
The heat capacity (C p ) is a fundamental thermodynamic property widely used to characterize biomolecular conformational stability. Despite extensive investigations into changes in the heat capacity (ΔC p ) associated with protein folding, these effects remain incompletely characterized in the folding of ribonucleic acid (RNA) molecules. In this work, we report on the thermophysical properties of a transfer RNA (tRNA) isodecoder in its chemically unmodified form as well as two modified forms, each having a distinct chemical modification, N 2,N 2-dimethylguanosine (m2,2G) or N 7-methylguanosine (m7G). The computed C p profile of the unmodified tRNA displayed a well-defined peak consistent with experimentally reported C p thermograms for various tRNAs. Based on the consistency between the computed thermophysical properties and experimental observations, we further probed the effects of different monovalent ions, distinct interatomic potentials, and varying melting temperature values on the key thermophysical properties of the tRNA isodecoder.
Lev Levintov, Esteban A. Orellana, Harish Vashisth· Journal of Physical Chemistr...· 0 citations
The vital functions of life are driven by proteins — long folded strings of amino acids. Millions of proteins exist in nature, all optimized for a certain set of operating conditions. One particularly important trait of a protein is thermostability, or the tendency to maintain form and function at high temperatures. Protein thermostability is important for both biological organisms and industrial enzymatic processes that utilize them. This work proposes a novel hybrid EC approach, employing the EAs Simulating Molecular Evolution (EASME) software, for evolving thermostable variants of proteins with minimal computational overhead and customized problem-specific fitness functions. A variation of NSGA-II with a novel recombination/mutation interplay was designed to tackle the specific challenges of working with proteins. The results of this work, which computationally improved the thermostability of a protein more effectively than a state of the art machine learning model from 2025, demonstrate that EC may have been historically undervalued in the realm of protein design. The pipeline designed for this work could be applied to numerous other proteins, easily expanded to optimize other traits of those proteins, or employed on another problem entirely where mutations have a notable fitness cost.
J. S. Browning, D. Tauritz, John Beckmann· Annual Conference on Genetic...· 0 citations
Related blog posts
MIT News · Artificial Intelligence· news.mit.eduAug 27, 2026
A new machine-learning framework aims to improve the success rate of computational protein design while moving away from results that reproduce sequences found in nature.