Electrostatic stabilization of a thermophilic cold shock protein.

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy.     

Red drum Sciaenops ocellatus mortalities associated with Streptococcus iniae infection.

We isolated for the first time Streptococcus iniae strains associated with diseased marine fish. Diseased red drum Sciaenops ocellatus were lethargic, and presented external signs (exophthalmia and loss of orientation) resembling those of freshwater fish infected by S. iniae. Skin lesions, extending to a necrotizing myositis, were typical of S. iniae infection of red drum. Histopathological findings indicate that S. iniae infection in red drum produces a chronic disease with systemic involvement characterized by multiple necrotic foci. Molecular epidemiology (RFLP [restriction fragment length polymorphism] ribotyping) revealed that 2 different ribotypes were involved in a single outbreak. The first is the EcoRI 'Israeli' trout and tilapine ribotype (Hind III type a strains), while the second is the EcoRI 'American' ribotype (Hind III type b strains), typical of tilapines farmed in Texas and Idaho.     

Pain.

Advances in our understanding of the activation of peripheral damage-sensing neurons (nociceptors) over the past year have been complemented by electrophysiological and imaging studies of central nervous system pain-related centres. The manipulation of gene expression in a reversible and cell type specific way combined with imaging and electrophysiological studies holds promise for helping us to identify the spatial and molecular substrates of pain perception with increasing precision and gives hope for improved analgesic therapies.     

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. © 1998 Wiley-Liss, Inc.