Protein stability thermostability

Littlechild JA, Guy J, Connelly S, Mallett L, Waddell S, Rye CA, Line K, Isupov M. Natural methods of protein stabilization thermostable biocatalysts. Biochem Soc Trans 2007 35 1558-1563. 

The term thermal stability (also thermostability) refers to the resistance of a protein to adverse intrinsic and extrinsic environmental influences, i.e., the thermal characteristic of the protein to remain steady against the dena-turation of its molecular integrity and inactivation of its biologic activity on facing high temperatures or other deleterious agents (6). One of the most important indices to measure protein stability is the decimal reduction time, or D-value, the time required to reduce 90% of the initial protein concentration exposed to the reference temperature. The D-value was used... 

One global property suitable for evaluation of evolutionary intermediates is protein stability. The unique biological and physical properties of an enzyme are highly dependent on the integrity of its native conformation. The structural stability of a protein can be measured by its resistance to heat, acid, and various chaotropic agents. Measurements of the thermostability of lysozymes provide an easy and an accurate way to assess the protein stability. 

Knowing that peptides and amines confer thermal stability on enzymes from certain thermophilic organisms (47-49) led some workers to examine protein stabilization by antibodies. It was found that the presence of specific polyclonal antibodies stabilize several enzymes (50, 51). In addition, not only did antibodies increjise the thermostability of a-amylase, glucoamylase, and subtilisin, but some stability toward acid denaturation, oxidizing agent, and organic solvent exposure was increased in specific cases (52, 53). 

Peptides that form a helices that associate as coiled coils, or as three- or four-helix tetrameric bundles or amphipathic helices that associate with lipid bilayers have been made. More difficult has been the design of proteins that form (3 sheets. Many efforts are being made to xmderstand protein stability " by systematic substitutions of one residue for another. Addition of new disulfide linkages at positions selected by study of three-dimensional structures sometimes stabilizes enzymes. On the other hand elimination of unnecessary cysteine residues can enhance stability by preventing p elimination and replacement of asparagine by threonine can improve the thermostability of enzymes by preventing deami-dation. ... 

The thermostability effect at each of the 35 selected glycosylation sites of SH3 is depicted in Figure 3a, and illustrates that glycosylation sites located on loops (less structured positions) are more effective in enhancing protein stability than other sites that are more structured. 

Figure 5 Folding characteristics of tailed proteins. The effects of the length of the attached flexible tail on the protein s thermostability (a) and the protein s radius of gyration in the unfolded state (the tails were attached to an SH3 domain at residue 36). The stability and radius of gyration changes are...

All enzymes are proteins, which are linear sequences of amino acids linked by peptide bonds. The folding of these sequences determined the secondary structure (such as a-helix, p-sheet or p-turn) and tertiary structure. Therefore, the properties of an en me are actually presumed from its sequence of amino acids. Some amino acids, dubbed hot spots , especially the ones in the active site where substrate binds, are sensitive to catalytic properties of an enzyme. Substitution of these important amino acids can significantly improve the activity or enantioselectivity toward a certain reaction. Protein stability is also maintained by the intramolecular and intermolecular interactions between residues of amino acids, including van der Waals forces, hydrophobic forces, electrostatic forces, hydrogen bonds and disulfide bonds. Detailed analysis of these amino acids, usually located in the protein surface, sheds light on the protein design for better thermostability. 

The application test of protein-engineered, thermostable glucose isomerase. The activity of immobilized enzyme is plotted as a function of time. The stability at 70 °C indicates how the enzymes will behave under industrial conditions. The variant Lys253Arg oi Actinoplanes missouriensis glucose isomerase has been shown to have (also under industrial conditions) a doubled half-life... 

We will discuss three different approaches to engineer a more thermostable protein than wild-type T4 lysozyme, namely (1) reducing the difference in entropy between folded and unfolded protein, which in practice means reducing the number of conformations in the unfolded state, (2) stabilizing tbe a helices, and (3) increasing the number of bydropbobic interactions in tbe interior core. 

Stability of several enzymes like proteases from thermophilic micro-organisms can be increased in aqueous-organic biphasic systems. Owusu and Cowan [67] observed a strong positive correlation between bacterial growth temperature, the thermostability of free protein extracts, and enzyme stability in aqueous-organic biphasic systems (Table 1). Enzymes, like other cell components (membranes, DNA, (RNA ribosomes), are adapted to withstand the environmental conditions under which the organism demonstrates optimal growth. 

Neupogen), a four-helix bundle cytokine, is formulated at pH 4 but has been shown to maintain both thermal stability and tertiary structure at pH 2.60 In fact, the secondary structure of this molecule was shown to remain highly helical at pH 4 (Tm approximately 62°C) and 2 (Tm approximately 63°C) as compared to pH 7 (Tm approximately 55°C) where a less conformationally stable form was observed. In the same study, FTIR and CD data corroborated the tendency of the protein to unfold as measured by the loss of helical structure in the order pH 7 > pH 4 > pH 2. Moreover, after determining optimal pH conditions of thermostability, several studies have shown that excipient screening at such conditions can successfully predict the rank of formulation cocktails that offer the most favorable stability.14 23 31 56... 

Further investigations revealed additional advantages conferred by the association of the partners in the T. thermophilus transamidosome. The kinetics of the hydrolysis of the aa-tRNAs in free form or bound in the complex show that the transamidosome stabilizes the ester bond of the Asp-tRNA intermediate by increasing its half-life about twofold (half-lives 315 and 204 min) and that of the end product Asn-tRNA fourfold (half-lives 18 and 66 min). Finally, measurements of the thermostability of the protein partners reveal a significant increase in the stability of GatCAB and the ND-AspRS at 85 °C, the optimal growth temperature of T. thermophilus, when associated in the transamidosome. 

Despite these numerous studies, it is still a matter of debate as to which structural factors are the main determinants of the increased stability in thermostable proteins. There are substantial differences between the... 

Table 1. List of the most relevant factors attributed to the greater stability of thermostable proteins...

Brazzein is another small sweet-tasting protein whose solution structure has been recently solved by NMR. Brazzein tastes 2000 times sweeter than sucrose on a weight basis and is exceptionally thermostable. As indicated by NMR, the structure of this 54 residue, single-chain polypeptide does not change between 32 and 82 °C and retains its sweetness after incubation at 98 °C for two hours.Brazzein contains one a-helix and three strands of antiparallel jd-sheet stabilized by four intramolecular disulphide bonds. It has been proposed that the disulphide bonds could be responsible for the thermostability of brazzein by forming a compact structure at the tertiary level.The structure of brazzein does not resemble that of the other two sweet proteins with known structures, monellin and thaumatin, whereas sequence alignment and structural prediction indicate that brazzein shares the fold of a newly identified family of serine proteinase inhibitors. 

Several proteins from different sources have been shown to maintain stability at high temperatures and NMR studies have been carried out in order to reveal their structures and/or to understand their activity. The most relevant references of a miscellany of thermostable proteins are reported in Table 3. Some of them such as bovine pancreatic trypsin inhibitor (BPTI), thermolysin and lysozyme have been widely studied as model systems in protein science. 

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