Protein thermostability

Matthews, B.W., Nicholson, H., Becktel, WJ. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc. Natl. 

Reetz, M.T., Carballeira, J.D. and Vogel, A. (2006) Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angewandte Chemie (International Edition in English), 45, 7745-7751. 

TlFd (59 amino acids, stable up to 90 °C) contains a single cluster that can exist in both 3Fe-4S and 4Fe-4S forms. The molecular and electronic structures were solved by NMR whereas the X-ray structure is still unknown. Compared to other mesophilic and thermophilic Fds, TTFd showed several structural adjustments such as the addition of a third strand of jS-sheet, a likely Lys2-Glu38 salt bridge from this /1-sheet and the N-terminus and a more hydrophobic and compact interaction between the large /S-sheet and the long helix. According to the authors, each of these modifications contributes to the extraordinary protein thermostability. 

Although not directly related to protein thermostability, several NMR studies have been also carried out on other Hsps such as mouse Hsp25, E. coli Hsp40 and mammalian Hsp70. ... 

Protein thermostability may be related to glycosylation and compactness but not cysteine disulfide bonds. 

To ascertain the upper limit of protein thermostability and to evaluate the effect of additional disulfide bridges on the enhancement of protein thermostability, additional cysteine residues were introduced into several unrelated proteins by site-directed mutagenesis and deactivation behavior tested at 100°C (Volkin, 1987). All the proteins investigated underwent heat-induced beta-elimination of cystine residues in the pH 4—8 range with first-order kinetics and similar deactivation constants kj that just depended on pH 0.8 0.3 h-1 at pH 8.0 and 0.06 0.02 h 1 at pH 6.0. These results indicate that beta-elimination is independent of both primary amino acid sequence and the presence of secondary structure elements. Elimination of disulfides produces free thiols that cause yet another deleterious reaction in proteins, heat-induced disulfide interchange, which can be much faster than beta-elimination. 

Thompson, M. J. and Eisenberg, D. (1999). Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability. J. Mol. Biol. 290, 595-604. 

Suzuki, Y. (1989). A general principle of increasing protein thermostability. Proc. Jpn. Acad., 65, 146-148. 

Various factors that contribute to protein thermostability, such as van der Waals interactions (17), core hydrophobicity (18-20), networks of hydrogen bonds (4, 5, 21), amount of secondary stmcture (4, 22), ionic interactions (6, 7, 23), packing density (24), and decreased length of surface loops (25), have been a subject of intense study for several decades. The major challenge, however, is to hnd out how the above factors are chosen and their combinations are formed by natural selection responding to the environmental temperature and depending on the evolutionary history of the organism (26). 

The first approach to simulation of protein thermophilic adaptation is to start from a purely statistical-mechanical analysis of protein thermostability. A specific Monte-Carlo procedure [the... 

Vetriani C, Maeder DL, Tolliday N, Yip KS, StUhnan TJ, Britton 42. KL, Rice DW, Klump HH, Robb FT. Protein thermostability above 100 degrees C a key role for ionic interactions. Proc. 

Seehger D, de Groot B (2010) Protein thermostability calculations using alchemical free energy simulations. Biophys J 98 2309-2316... 

Baker SN, McQesky TM, Pandey S et al (2004) Fluorescence studies of protein thermostability in ionic liquids. Chem Commun (8) 940-941... 

Proteins found in hyperthermophiles display an astonishing resistance to thermal denaturation. Some are stable for hours or even days at temperatures near to the boiling point. This has attracted much attention as these proteins are promising candidates themselves as stable biocatalysts, and also provide valuable hints to the understanding of the mechanisms of protein thermostability. 

As the importance of ion-pairs toward protein thermostability has been stressed in many cases, addition or removal of an ion-pair should have significant effects. A clear example is provided by mutagenesis studies of glutamate dehydrogenase from T kodakaraensis KOD1 (Tk-GDH)[361. The GDH from Pyrococcus Juriosus (Pf-GDH) and Tk-GDH are 83% identical in terms of primary structure. However, while Pf GDH displays a half-life of 12 h at 100 C, that of Tk-GDH is 4 h. The three-... 

The examples mentioned above have shown that in many cases, sequence comparisons between two homologous enzymes with different thermostabilities provide valuable clues as to the how to increase protein thermostability rationally. An interesting observation has recently been made that even a set of amino acid sequences of homologous, mesophilic enzymes provides sufficient information to... 

The examples above represent some of the most successful studies in protein engineering. They show that it is possible to enhance protein thermostability rationally, alter cofactor or substrate specificity, regiospecificity, and even change catalytic activity. Furtherm ore, the creation of enzymatic activity from a non-catalytic protein backbone, and the creation of a biocatalyst with an unprecedented catalytic activity not found in nature, have also been achieved. However, the examples published in the literature are probably only a tiny fraction of the many studies that have been, or are still, in progress awaiting positive results. 

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