Computational investigation of cold denaturation in the Trp-cage miniprotein

Abstract

The functional native states of globular proteins become unstable at low temperatures, resulting in cold unfolding and impairment of normal biological function. Fundamental understanding of this phenomenon is essential to rationalizing the evolution of freeze-tolerant organisms and developing improved strategies for long-term preservation of biological materials. We present fully atomistic simulations of cold denaturation of an α-helical protein, the widely studied Trp-cage miniprotein. In contrast to the significant destabilization of the folded structure at high temperatures, Trp-cage cold denatures at 210 K into a compact, partially folded state; major elements of the secondary structure, including the α-helix, are conserved, but the salt bridge between aspartic acid and arginine is lost. The stability of Trp-cage's α-helix at low temperatures suggests a possible evolutionary explanation for the prevalence of such structures in antifreeze peptides produced by coldweather species, such as Arctic char. Although the 3 10 -helix is observed at cold conditions, its position is shifted toward Trp-cage's C-terminus. This shift is accompanied by intrusion of water into Trp-cage's interior and the hydration of buried hydrophobic residues. However, our calculations also show that the dominant contribution to the favorable energetics of low-temperature unfolding of Trp-cage comes from the hydration of hydrophilic residues.cold denaturation | Trp-cage miniprotein | protein folding T he functional native states of globular proteins that are stable near physiological conditions become labile when changes in temperature, pressure, and solvent composition alter their environment. The partial or complete unfolding of secondary and tertiary structure associated with this loss of stability can strongly affect protein behavior, leading to significantly impaired biological function (1, 2). Denaturation upon heating is a ubiquitous and well-studied phenomenon in which proteins gain configurational entropy and unfold as a result of increased kinetic energy. By contrast, the mechanisms responsible for pressure-induced unfolding, and for denaturation of globular proteins at low temperatures, remain incompletely understood (3-5).Fundamental understanding of cold denaturation is important due to its ecological implications and relevance to industrial processing of proteins and biological materials. Freeze-tolerant organisms such as the Arctic char, for example, thrive in subfreezing habitats where cold denaturation can occur (6). Biopharmaceuticals are also exposed to cold conditions that can result in denaturation when they are lyophilized into freeze-dried solids to prolong their shelf life (7,8). Natural cryoprotectants such as sugars and polyols stabilize proteins against denaturation in cold-weather species (6), and similar compounds have been used to mitigate the damaging effects of freeze-drying in pharmaceutical formulations (7, 9).Cold denaturation was first reported by Hopkins in 1930 (10). Brandts (11) subsequently observed ...

Cryo-Cooling Effect on DHFR Crystal Studied by Replica-Exchange Molecular Dynamics Simulations

“…7). This is consistent with (20), in which it is suggested that the hydrophilic residues make increased hydrogen bonds with water molecules at lower temperature, which is counted as a major driving force of cold denature.…”

“…9), suggesting that at low temperatures, the fluctuation of biological importance is hindered. In the crystal structure of apo form studied at room temperature at a resolution of 2.3 Å (PDB: 5DFR), some residues of Met20 loop (residues [16][17][18][19][20] were missing, and others (residues 21-24) were weak or partially disordered with high B factor (40-50 Å 2 ) (31), suggesting a large fluctuation in accord with our simulations. The conformational variations are visualized in Fig.…”

“…7) suggest that at lower temperatures, the solvation of hydrophilic residues increased much more than the solvation of hydrophobic residues. Because those observations can be seen as consistent with (20), the driving force of conformational effect by cryo-cooling should be the increase in interaction between hydrophilic residues and water molecules.…”

“…Classical MD simulation often suffers from insufficient sampling, particularly for large systems. The sampling issue can be alleviated by the replicaexchange MD (REMD) method (14)(15)(16)(17)(18), which has been successfully applied to Trp-cage (19) to study cold denaturation (20). Simulations of the crystal environment, in which a unit cell is built and periodic boundary conditions are applied in accordance with translational symmetry of the crystal concerned, have been reported (21)(22)(23)(24)(25)(26)(27)(28)(29).…”

Role of Water in the Selection of Stable Proteins at Ambient and Extreme Thermodynamic Conditions

“…As a matter of fact, many life forms survive under extreme T-P conditions [1,2], implying that their proteins fit that environment. Recent works [3][4][5][6][7][8][9][10] have evidenced that changes in T and P significantly alter the water-mediated hydrophilic and hydrophobic interactions of the residues along the chain, with an effect that depends on the sequence. However, a direct observation of how the protein selection responds to extreme changes in the aqueous conditions is still lacking.…”