Characterization of serum immunoglobulin M of the Antarctic teleost Trematomus bernacchii

Trematomus bernacchii immunoglobulin M concentration was determined in the serum by ELISA; the mean concentration value was 2.7 mg/ml corresponding to 9.6% of the total serum proteins. Purified IgM was analyzed by SDS-polyacrylamide gel electrophoresis, isoelectrofocusing and 2D electrophoresis. The relative molecular mass of the polymeric form was 830 kDa; that of separated H and L chains was, respectively, 78 and 25 kDa. The isoelectric points of the entire molecule ranged from 4.4 to 6.5, that of isolated H chains was between 4.0 and 6.0. Separated H chains were shown to reaggregate in tetrameric form. The cleavage site of trypsin was at the end of the CH1 domain, as confirmed by the N-terminal amino acid sequence of one of the resultant peptides. Immunoblotting was used to detect carbohydrates in the H and L chains labeled with digoxigenin. Glycosyl residues were detected only in the H chain. The carbohydrate content was evaluated to be 12.8% of the entire chain. Purified Igs were hydrolyzed by N-glycosidase F at different conditions and at least four different hydrolytic sites were revealed by limited deglycosylation. T. bernacchii IgM was also compared to those of five other polar fish species.     

Physics and biophysics of solvent induced forces: hydrophobic interactions and context-dependent hydration

Solvent induced forces (SIFs) among solutes derive from solvent structural modification due to solutes, and consequent thermodynamic drive towards minimization of related free energy costs. The role of SIFs in biomolecular conformation and function is appreciated by observing that typical SIF values fall within the 20–200 pN interval, and that proteins are stable by only a few kcal mol^–1 (1 kcal mol^–1 corresponds to 70 pN Å). Here we study SIFs, in systems of increasing complexity, using Molecular Dynamics (MD) simulations which give time- and space-resolved details on the biologically significant scale of single protein residues and sidechains. Of particular biological relevance among our results are a strong modulability of hydrophobic SIFs by electric charges and the dependence of this modulability upon charge sign. More generally, the present results extend our understanding of the recently reported strong context-dependence of SIFs and the related potential of mean force (PMF). This context-dependence can be strong enough to propagate (by relay action) along a composite solute, and to reverse SIFs acting on a given element, relative to expectations based on its specific character (hydrophobic/ philic, charged). High specificity such as that of SIFs highlighted by the present results is of course central to biological function. Biological implications of the present results cover issues such as biomolecular functional interactions and folding (including chaperoning and pathological conformational changes), coagulation, molecular recognition, effects of phosphorylation and more.     

Biomolecular-solvent stereodynamic coupling probed by deuteration

Thermodynamic interpretation of experiments with isotopically perturbed solvent supports the view that solvent stereodynamics is directly relevant to thermodynamic stability of biomolecules. According with the current understanding of the structure of the aqueous solvent, in any stereodynamic configuration of the latter, connectivity pathways are identifiable for their topologic and order properties. Perturbing the solvent by isotopic substitution or, e.g., by addition of co-solvents, can therefore be viewed as reinforcing or otherwise perturbing these topologic structures. This microscopic model readily visualizes thermodynamic interpretation. In conclusion, the topologic stereodynamic structures of connectivity pathways in the solvent, as modified by interaction with solutes, acquire a specific thermodynamic and biological significance, and the problem of thermodynamic and functional stability of biomolecules is seen in its full pertinent phase space.