Mesophile versus Thermophile: Insights Into the Structural Mechanisms of Kinetic Stability (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.
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.
PLoS Computational Biology, 2010
Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease a-lytic protease (aLP) has done just that, as its unfolding is both very slow (t 1/2 ,1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to aLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the aLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both aLP and trypsin. The simulated aLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that aLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of aLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases.
Protein Science, 2004
Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and ␣-lytic protease (␣LP) are synthesized with covalently attached pro regions necessary for their folding. In this article, we characterize the folding free energy landscape of SGPB and compare it to the folding landscapes of ␣LP and trypsin, a mammalian homolog that folds independently of its zymogen peptide. In contrast to the thermodynamically stable native state of trypsin, SGPB and ␣LP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and ␣LP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4-8 in protease resistance is accompanied by a cost of ∼10 5 in the spontaneous folding rate and ∼5-9 kcal/mole in thermodynamic stability. These penalties have been overcome by the coevolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.
Increasing Temperature Accelerates Protein Unfolding Without Changing the Pathway of Unfolding
Journal of Molecular Biology, 2002
We have traditionally relied on extremely elevated temperatures (498 K, 225 8C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298 K to 498 K. The simulation lengths vary from 94 ns to 20 ns, for a total simulation time of 344 ns, or 0.34 ms. At 298 K, the protein is very stable over the full 50 ns simulation. At 348 K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25 ns, explores partially unfolded conformations for 20 ns, and then refolds over the last 35 ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498 K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.
Characterization of the Protein Unfolding Processes
Correct folding is critical for the biological activities of proteins. As a contribution to a better understanding of the protein (un)folding problem, we studied the effect of temperature and of urea on peptostreptococcal Protein L destructuration. We performed standard molecular dynamics simulations at 300 K, 350 K, 400 K, and 480 K, both in 10 M urea and in water. Protein L followed at least two alternative unfolding pathways. Urea caused the loss of secondary structure acting preferentially on the b-sheets, while leaving the a-helices almost intact; on the contrary, high temperature preserved the b-sheets and led to a complete loss of the a-helices. These data suggest that urea and high temperature act through different unfolding mechanisms, and protein secondary motives reveal a differential sensitivity to various denaturant treatments. As further validation of our results, replicaexchange molecular dynamics simulations of the temperature-induced unfolding process in the presence of urea were performed. This set of simulations allowed us to compute the thermodynamical parameters of the process and confirmed that, in the configurational space of Protein L unfolding, both of the above pathways are accessible, although to a different relative extent.
A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.