Physical Principles Of Biomolecular Recognition

点击翻译按钮获取中文摘要

Interactions between various biological helices control protein folding and assembly, DNA packing, protein-DNA interactions, connective tissue formation and stability, and many other processes responsible for normal function and pathology in living organisms. By combining several experimental techniques (UV-VIS, fluorescence, CD and FTIR spectroscopy, x-ray diffraction, calorimetry, etc.) with rigorous physical theories, we continued to advance our understanding of these most basic molecular recognition reactions. Our most significant achievements during the past year were: (1) By combining ultra-slow scanning calorimetry and isothermal circular dichroism we found that at physiological conditions human lung collagen monomers denature within a couple of days and rat tail tendon collagen monomers denature within hours. Contrary to the wide-held belief, the energetically preferred conformation of monomeric collagen at body temperature is a random coil rather than a triple helix. Our data suggest that once secreted from cells collagen helices begin to unfold. Initial micro-unfolding of their least stable domains triggers self-assembly of fibers where the helices are protected from complete unfolding. Apparently, Nature adjusts collagen hydroxyproline content to ensure that the melting temperature of triple helical monomers is several degrees below rather than above body temperature. (2) We further characterized the effect of an insertion of a Gly-Ala-Hyp triplet near C terminal in a1(I) chain in a lethal human OI, an unusual mutation recently discovered by the group of Dr. Marini (HDB/NICHD). By comparing the kinetics of N-propeptide cleavage by type I collagen N-protease, we demonstrated that the insertion leads to a register shift along the whole length of the triple helix rather than "looping out" of the tripeptide. The register shift causes a conformational change in the N-propetide (some 850 residues away from the mutation site), a change in the recognition of the propeptide by N-protease and a change in the specific cleavage kinetics. (3) We determined the extent of posttranslational overmodification of collagen in different tissues from Brtl IV mouse model of osteogenesis imperfecta developed by the group of Dr. Marini. Our present data indicate that the overmodification may not be a significant factor in phenotype variation observed in these mice. (4) We developed a new difference gel electrophoresis technique for traditional protein gels. The technique is based on fluorescent labeling of proteins by different dyes so that differently labeled proteins can be mixed together for further analysis. It is particularly useful for analysis of small differences in the molecular weight (e.g., posttranslational overmodification) and comparison of enzymatic processing of wild type and mutant proteins by co-processing differently labeled proteins in the same test tube. (4) We developed a theory relating the microscopic physics of helix-helix interaction to macroscopic properties of cholesteric DNA assemblies. Estimates based on this theory rationalized the large value of the observed cholesteric pitch and its nonmonotonic dependence on the spacing between DNA molecules.