Physical Principles Of Biomolecular Recognition, Self-as

Child Health And Human Development

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Abstract

Section on Physical Biochemistry, OD/NICHD conducts experimental and theoretical studies of structure and function of biomolecules with emphasis on molecular mechanisms of pathology in connective tissue disorders. Through collaboration with clinical researchers, we strive to gain better knowledge and develop novel techniques for diagnostics, characterization and treatment of osteogenesis imperfecta (OI) and other diseases. Over the years we reported first direct measurements and established physical nature of forces between collagen molecules. We discovered that both procollagen and collagen are intrinsically unstable at physiological conditions so that cells have to use molecular chaperones to fold procollagen within the Endoplasmic Reticulum (ER). We found that micro-unfolding of most thermally labile regions of collagen triple helix is necessary for proper molecular recognition and fiber formation. In fibers, collagen helices are protected from complete unfolding but they constantly undergo transient local unfolding and refolding giving the fibers their unique combination of elasticity and strength. In the last few years the research focus of our group shifted from fundamental studies of these processes to understanding how different OI mutations affect them. In particular, during the last year we extended our collaboration with BEMB/NICHD scientists on studies of physical and chemical properties of mutant collagens from OI patients. We confirmed posttranslational overmodification of type I collagen in several new BEMB patients who were diagnosed with OI but were found to have no type I collagen mutations. The search for the source of this posttranslational overmodification is presently focused on possible defects in molecular chaperones needed for procollagen folding. We discovered structural changes in type I collagen from one long-term BEMB patient, confirming the presence of a mutation not found before. This work prompted BEMB scientists to start screening for more mutations, which could have been missed in initial sequencing. We continued systematic analysis of physical and chemical properties of collagen from the collection of other long-term BEMB patients whose mutations are known. Chartacterization of over a dozen additional mutant collagens confirmed our previous hypothesis that changes in the triple helix stability caused by substitutions of obligatory Gly residues depend primarily on the position of the mutation within certain domains rather than on the the identity of the substituting residue or its immediate local environment. In collaboration with scientists from University of Pavia, Italy and BEMB/NICHD we continued to study the unusual rescue of lethal OI phenotype and moderation of OI symptoms in homozygous mice with dominant G349C substitution in alpha1(I) chain of type I collagen. We found that tissues from heterozygous animals contain substantially smaller amount of molecules with a single mutant chain than expected from known expression level of the mutant allele. Our study of collagen secretion in cell culture revealed that dermal fibroblasts selectively retain and degrade a significant fraction of molecules containing both mutant and normal alpha1(I) chains, while molecules with both mutant chains clear the secretory pathway at almost normal rate. The observed accumulation of unsecreted molecules is likely to cause additional ER stress in collagen producing cells of heterozygous animals and make these cells less viable. Substantially better secretion of molecules containing only mutant alpha1(I) chains is, therefore, likely to improve the viability of fibroblasts and osteoblasts in homozygous animals, potentially explaining their less severe OI phenotype. Additional experiments designed to verify this hypothesis are currently under way. Another important direction of our research is closely related recognition and assembly reactions involviingg DNA. In particular, we uncovered several common physical principles, which govern formation, structure and physical properties of collagen and DNA aggregates. We suggested mechanisms for counter-ion specificity in DNA condensation, DNA overwinding from 10.5 base pairs per helical turn in solution to 10.0 bp/turn in aggregates, sequence homology recognition in pairing of duplex DNA and several other phenomena. The present focus of these studies is measurement of sequence effects in formation, structure and properties of DNA aggregates. During the last year, we concentrated on attempts to develop a theory necessary for extracting information on mutal azimuthal alignment of adjacent helices from x-ray diffraction patterns of highly oriented DNA samples. Preliminary analysis of x-ray diffraction data revealed significant biaxial correlations between DNA molecules even at 15 to 20 Angstrom surface-to-surface separation between DNA helices, confirming our previous theoretical estimates and validating the assumptions built into our theory of sequence-dependent electrostatic interactions between DNA.