Metastable Water Clusters in the Nonpolar Cavities of the Thermostable Protein Tetrabrachion (original) (raw)
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Journal of Molecular Biology, 2005
a-Lytic protease (aLP) and Streptomyces griseus protease B (SGPB) are two extracellular serine proteases whose folding is absolutely dependent on the existence of their companion pro regions. Moreover, the native states of these proteins are, at best, marginally stable, with the apparent stability resulting from being kinetically trapped in the native state by large barriers to unfolding. Here, in an effort to understand the physical properties that distinguish kinetically and thermodynamically stable proteins, we study the temperature-dependences of the folding and unfolding kinetics of aLP and SGPB without their pro regions, and compare their behavior to a comprehensive set of other proteins. For the folding activation thermodynamics, we find some remarkable universal behaviors in the thermodynamically stable proteins that are violated dramatically by aLP. Despite significant variations in DC ‡ P;F , the maximal folding speed occurs within the narrow biological temperature range for all proteins, except for aLP, with its maximal folding speed shifted lower by 200 K. This implies evolutionary pressures on folding speed for typical proteins, but not for aLP. In addition, the folding free energy barrier in the biological temperature range for most proteins is predominantly enthalpic, but purely entropic for aLP. The unfolding of aLP and SGPB is distinguished by three properties: a remarkably large DC ‡ P;U , a very high DG ‡ U , and a maximum DG u ‡ at the optimal growth temperature for the organism. While other proteins display each of these traits to some approximation, the simultaneous optimization of all three occurs only in the kinetically stable proteins, and appears to be required to maximize their unfolding cooperativity, by suppressing local unfolding events, and slowing the rate of global unfolding. Together, these properties extend the lifetime of these enzymes in the highly proteolytic extracellular environment. Attaining such functional properties seems 0022-2836/$ -see front matter q , maximum value of DG U ‡ with temperature; T S,U ‡ , temperature at which KTDS U ‡ crosses 0; DG F,min ‡ , minimum value of DG F ‡ with temperature; T S,F ‡ , temperature at which KTDS F ‡ crosses 0; ACP, human muscle acylphosphatase; a-specSH3, PWT variant of a-spectrin Src homology region 3 (SH3) domain with second and third residues substituted for a Gly residue; CD2.d1, domain 1 (residues 1-98) of T-cell adhesion protein CD2; CI2, chymotrypsin inhibitor 2; Bc CspB, Bacillus caldolyticus cold-shock protein; Bs CspB, Bacillus subtilis cold-shock protein; FKBP, human FK506 binding protein 12; Hpr, Escherichia coli histidine-containing phosphocarrier protein; N-PGK, Bacillus stearothermophilus N-terminal domain (residues 1-175) of phosphoglycerate kinase; NTL9, Bacillus stearothermophilus N-terminal domain (residues 1-56) of L9; protein L, Y43W point mutant of protein L; tendamistat, Streptomyces tendae a-amylase inhibitor tendamistat; y. isocyto-c, Saccharomyces cerevisiae iso-2 cytochrome c. possible only through the gross perturbation of the folding thermodynamics, which in turn has required the co-evolution of pro regions as folding catalysts.
Thermal breaking of spanning water networks in the hydration shell of proteins
The Journal of Chemical Physics, 2005
The presence of a spanning hydrogen-bonded network of water at the surface of biomolecules is important for their conformational stability, dynamics, and function. We have studied by computer simulations the clustering and percolation of water in the hydration shell of a small elastinlike peptide ͑ELP͒ and the medium-size protein staphylococcal nuclease ͑SNase͒, in aqueous solution. We have found that in both systems a spanning network of hydration water exists at low temperatures and breaks up with increasing temperature via a quasi-two-dimensional percolation transition. The thermal breaking of the spanning water network occurs at biologically relevant temperatures, in the temperature range, which is close to the temperature of the "inverse temperature transition" of ELP and the unfolding temperature of SNase, respectively.
The Journal of Physical Chemistry B, 2009
Molecular motions and properties of water and biomacromolecules change when confined in nanoporous matrices. Freezing and melting points of water are depressed, and generally, the activity of enzymes and stability of proteins are increased. We performed temperature ramp FTIR analyses of silica matrix confined water and proteins to identify the kinetic and thermodynamic transitions of water at cryogenic temperatures and to understand the water-protein interactions in confinement. In our studies, confined water did not freeze at temperatures as low as -180°C but underwent liquid-liquid and liquid-glass transitions during cooling. During warming from cryogenic temperatures, the formations of cubic and hexagonal ice were detected. Additionally, the changes in the secondary structures of proteins correlated to the changes in the H-bonding characteristics of the confined water. Our results showed that the kinetic and thermodynamic transitions of water dictate the structural transitions of encapsulated proteins. Evidence was obtained for the universal behavior of water in close proximity to surfaces and in the hydration shells of isolated and cytoplasmic proteins (in intact encapsulated bacteria and mammalian cells).
Thermodynamical Implications of a Protein Model with Water Interactions
Journal of Theoretical Biology, 2001
We refine a protein model that reproduces fundamental aspects of protein thermodynamics. The model exhibits two transitions, hot and cold unfolding. The number of relevant parameters is reduced to three: 1) binding energy of folding relative to the orientational energy of bound water, 2) ratio of degrees of freedom between the folded and unfolded protein chain and 3) the number of water molecules that can access the hydrophobic parts of the protein interior. By increasing the number of water molecules in the model, the separation between the two peaks in the heat capacity curve comes closer, which is more consistent with experimental data. In the end we show that if we, as a speculative assumption, assign only two distinct energy levels for the bound water molecules, we obtain better correspondence with experiments.
Thermodynamics of the temperature-induced unfolding of globular proteins
Protein Science, 1995
The heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature-induced unfolding of 11 globular proteins of known three-dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent-accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 "C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of nonpolar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature-induced unfolding. J Mol BiOl 224:715-723. 110:537-568. &on. FEBS Lett 9157-58. . Weaver LH. Gray TM. Gruetter MG, Anderson DE, Wozniak JA, Dahlquist sensitive mutant of phage lysozyme, Arg 96 + His. Biochernisrry 28: FW, Matthews BW. 1989. High-resolution structure of the temperature-3793-3797. Wlodawer A, Deisenhofer J, Huber R. 1987. Comparison of two highlyrefined structures of bovine pancreatic trypsin inhibitor. J Mol Biol /93:145-156.
Langmuir : the ACS journal of surfaces and colloids, 2016
Proteins from organisms which have adapted to environmental extremes provide excellent model systems to determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on hyperthermophilic protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal β-strand (TmCSP V62A). We use M...
Probing the Unfolding Region in a Thermolysin-like Protease by Site-Specific Immobilization †
Biochemistry, 1999
Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysinlike protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9-and 12-fold increase in half-lives at 75°C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLPste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.
Water Contributes Actively to the Rapid Crossing of a Protein Unfolding Barrier
Journal of Molecular Biology, 2002
The cold-shock protein CspB folds rapidly in a N Y U two-state reaction via a transition state that is about 90% native in its interactions with denaturants and water. This suggested that the energy barrier to unfolding is overcome by processes occurring in the protein itself, rather than in the solvent. Nevertheless, CspB unfolding depends on the solvent viscosity. We determined the activation volumes of unfolding and refolding by pressure-jump and high-pressure stopped-flow techniques in the presence of various denaturants. The results obtained by these methods agree well. The activation volume of unfolding is positive ðDV ‡ NU ¼ 16ð^4Þ ml=molÞ and virtually independent of the nature and the concentration of the denaturant. We suggest that in the transition state the protein is expanded and water molecules start to invade the hydrophobic core. They have, however, not yet established favorable interactions to compensate for the loss of intra-protein interactions. The activation volume of refolding is positive as well ðDV ‡ UN ¼ 53ð^6Þ ml=molÞ and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, DV ‡ UN decreases significantly, suggesting that compact unfolded forms become populated under these conditions. Abbreviations used: CspB, cold-shock protein from Bacillus subtilis; GdmSCN, guanidinium thiocyanate; l, measured rate of a reaction; k ij , microscopic rate constant; DV ‡ app , apparent activation volume; DV ‡ ij , microscopic activation volume; n, n ij , denaturant-dependence of reaction and activation volumes.
Mesophile versus Thermophile: Insights Into the Structural Mechanisms of Kinetic Stability
Journal of Molecular Biology, 2007
Obtaining detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. Comparisons between proteins adapted to survive extreme temperatures with their mesophilic homologs are likely to provide valuable information on the interactions relevant to the unfolding transition. For kinetically stable proteins such as α-lytic protease (αLP) and its family members, their large free energy barrier to unfolding is central to their biological function. To gain new insights into the mechanisms that underlie kinetic stability, we have determined the structure and high temperature unfolding kinetics of a thermophilic homolog, Thermobifida fusca protease A (TFPA). These studies led to the identification of a specific structural element bridging the N and Cterminal domains of the protease (the "domain bridge") proposed to be associated with the enhanced high temperature kinetic stability in TFPA. Mutagenesis experiments exchanging the TFPA domain bridge into αLP validate this hypothesis and illustrate key structural details that contribute to TFPA's increased kinetic thermostability. These results lead to an updated model for the unfolding transition state structure for this important class of proteases in which domain bridge undocking and unfolding occurs at or before the TS. The domain bridge appears to be a structural element that can modulate the degree of kinetic stability of the different members of this class of proteases.