Mutants thermodynamic stability

Figure B3.5.12 Effect of mutations detected by CD. The far-UV CD spectra (A) show that the secondary structure of p-lactamase PC1 (solid line) from Staphylococcus aureus is essentially unaffected by point mutations P2 (Thr 140—>lle dashed line) and P54 (Asp 146->Asn dotted line). The crystallographic structure of P54 (Herzberget al., 1991) confirms that, apart from a loop region, the main body of the molecule that contains the thirteen tyrosine residues is very closely similar to that in the wild-type enzyme. The intensity of the tyrosine ellipticity (B) is, however, markedly decreased in each of the mutants, the lower thermodynamic stabilities of which support the interpretation of increased dynamics (Craig et al., 1985).

K. D. Wittrup. Secretion effidency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability. Biochemistry (1998) 37(5) 1264-73. 

The specific activity and thermodynamic stability of the (C2A, ClOA) mutant confirm that the Cys-2 to Cys-10 disulfide bond imparts thermodynamic stability but has litde effect on catalytic activity. Hence this mutant was selected as the starting point for constructing a circularly permuted form of RNase-Tl so that as short a linker as possible could be used to bridge the original N- and C-termini. The activity and stability of the circularly permuted variant indicate that it adopts an overall tertiary fold very similar to that of the native protein. Therefore, transposing the first 34 residues to the C-terminus has little effect on the overall folding to the final tertiary structure. The real effect, however, may be more evident in the kinetics of the specific folding pathway. 

Thermodynamic stability is a global property of the enzyme structure, and contributions of individual amino acids toward the free energy of folding are additive and highly cooperative. Analysis of mutant proteins has defined the contributions of various amino acids toward the overall stability of the protein. Replacements which alter the formation of ion pairs, hydrogen bonds, van der Waals contacts, or hydrophobic interactions each tend to destabilize the folded protein by a qualitatively comparable amount (14). In one approach to this problem, site-specific substitution of amino acids has provided new approaches toward dissecting the kinetic mechanisms of protein folding (27). 

In view of the presumed lower thermodynamical stability of replacement folds compared to the A-B fold, crosses of petites 14 and 26 with wild-type cells were also performed at both a higher (33°C) and a lower (23°C) temperature than the standard one, 28°C. The results of Table 2.2 indicate that such temperatures decreased and increased, respectively, the replicative ability of the two mutants, whereas that of the control orT petite Z1 did not show any significant change between these temperatures. The decrease of the replicative ability was stronger for petite 14 than for petite 26, as expected from the presumably lower thermodynamical stability of the replacement fold of the former. In the first case, the proportion of wild-type colonies obtained at 23°C and 33°C, respectively, was 10% and 55%, in the second 11% and 42%. The changes in replicative ability just described are immediate and reversible. 

Singh and Kollman reported on the first FEP calculations conducted to investigate the thermodynamic stability of mutant RNA hairpins. They were able to attribute the increased observed stability of an UUCG over an UUUG loop to a phosphate-base interaction which favors cytosine over uracil by 6 kcal mor. MD simulations on RNA/DNA hybrids have been conducted by Cheatham et al, ... 

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

Abstract. Walter Kauzmann stated in a review of protein thermodynamics that volume and enthalpy changes are equally fundamental properties of the unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behaviour (Nature 325 763-764, 1987). While the thermodynamic basis for pressure effects has been known for some time, the molecular mechanisms have remained rather mysterious. We, and others in the rather small field of pressure effects on protein structure and stability, have attempted since that time to clarify the molecular and physical basis for the changes in volume that accompany protein conformational transitions, and hence to explain pressure effects on proteins. The combination of many years of work on a model system, staphylococcal nuclease and its large numbers of site-specific mutants, and the rather new pressure perturbation calorimetry approach has provided for the first time a fundamental qualitative understanding of AV of unfolding, the quantitative basis of which remains the goal of current work. 

ProTherm (16) is a large collection of thermodynamic data on protein stability, which has information on 1) protein sequence and stmcture (2) mutation details (wild-type and mutant amino acid hydrophobic to polar, charged to hydrophobic, aliphatic to aromatic, etc.), 3) thermodynamic data obtained from thermal and chemical denaturation experiments (free energy change, transition temperature, enthalpy change, heat capacity change, etc.), 4) experimental methods and conditions (pH, temperature, buffer and ions, measurement and method, etc.), 5) functionality (enzyme activity, binding constants, etc.), and 6) literature. 

---