Dieter Perl

Two exposed amino acid residues confer thermostability on a cold shock protein

Thermophilic organisms produce proteins of exceptional stability. To understand protein thermostability at the molecular level we studied a pair of cold shock proteins, one of mesophilic and one of thermophilic origin, by systematic mutagenesis. Although the two proteins differ in sequence at 12 positions, two surface-exposed residues are responsible for the increase in stability of the thermophilic protein (by 15.8 kJ mol−1 at 70 °C). 11.5 kJ mol−1 originate from a predominantly electrostatic contribution of Arg 3 and 5.2 kJ mol−1 from hydrophobic interactions of Leu 66 at the carboxy terminus. The mesophilic protein could be converted to a highly thermostable form by changing the Glu residues at positions 3 and 66 to Arg and Leu, respectively. The variation of surface residues may thus provide a simple and powerful approach for increasing the thermostability of a protein.

The effects of ionic strength on protein stability: the cold shock protein family.

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.

THE FAMILY OF COLD SHOCK PROTEINS OF BACILLUS SUBTILIS : STABILITY AND DYNAMICS IN VITRO AND IN VIVO

Bacillus subtilis possesses three homologous small cold shock proteins (CSPs; CspB, CspC, CspD, sequence identity >72%). They share a similar β-sheet structure, as shown by circular dichroism, and have a very low conformational stability, with CspC being the least stable. Similar to CspB, CspC and CspD unfold and refold extremely fast in a N ⇌ U two-state reaction with average lifetimes of only 100–150 ms for the native state and 1–6 ms for the unfolded states at 25 °C. As a consequence of their low stability and low kinetic protection against unfolding, all three cold shock proteins are rapidly degraded by proteases in vitro. Analysis of the CSP stabilitiesin vivo by pulse-chase experiments revealed that CspB and CspD are stable during logarithmic growth at 37 °C as well as after cold shock. The cellular half-life of CspC is shortened at 37 °C, but under cold shock conditions CspC becomes stable. The proteolytic susceptibility of the CSPs in vitro was strongly reduced in the presence of a nucleic acid ligand, suggesting that the observed stabilization of CSPs in vivo is mediated by binding to their substrate mRNA at 37 °C and, in particular, under cold shock conditions.

Surface-exposed phenylalanines in the RNP1/RNP2 motif stabilize the cold-shock protein CspB from Bacillus subtilis

In the cold‐shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single‐stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea‐induced and heat‐induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing. Proteins 30:401–406, 1998.

Water contributes actively to the rapid crossing of a protein unfolding barrier

The cold-shock protein CspB folds rapidly in a N <= => 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 (Delta V(++)(NU)=16(+/-4) ml/mol) 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 (Delta V(++)(NU)=53(+/-6) ml/mol) and, above 3 M urea, independent of the concentration of the denaturant. At low concentrations of urea or guanidinium thiocyanate, Delta V(++)(UN) decreases significantly, suggesting that compact unfolded forms become populated under these conditions.

Some like it hot: The molecular determinants of protein thermostability

Extremophilic organisms survive under harsh environmental conditions. They prefer to live at high hydrostatic pressure, in the presence of high salt concentrations, in acid or alkaline solutions, and, in particular, at high temperature. The thermophiles and hyperthermophiles, some of which can grow beyond 110 8C, receive much attention, because their thermostable proteins have become important tools in biochemistry and industrial biotechnology. At the same time they provide us with the opportunity to elucidate the origins of protein stability, to learn how thermostability is encoded in the amino acid sequence, and, ultimately, to use this information for designing stable proteins. 3] Mesophilic organisms are not forced to maintain or evolve thermostable constituents and, as a consequence, most of their proteins show very low conformational stabilities. This may have a simple reason. The overwhelming majority of the mutations, as they occur during evolution, are disadvantageous for stability and function, and therefore the stability of a protein is maintained just high enough to secure its proper function in the organism. Proteins are stabilized primarily by noncovalent interactions, such as hydrogen bonds, hydrophobic interactions, or coulombic forces. There is a multitude of interactions in proteins, but all of them are weak, they must balance the loss of interactions with the aqueous solvent and, in particular, they must compensate for the enormous decrease in chain entropy upon folding. Makhatadze and Privalov estimate a decrease in chain entropy equivalent to TDS ÿ1500 kJ molÿ1 upon folding a protein with 100 residues. It is thus not surprising that most proteins are only marginally stable, with Gibbs free energies of denaturation (DGD) often in the range of 10 ± 60 kJ molÿ1. The finely tuned balance between many stabilizing and many destabilizing interactions complicates the analysis, and it remains difficult to identify the molecular origins of the extra stability of the thermophilic proteins.