The understanding of the interaction of membranes proteins with membranes is hampered by our lack of physical techniques to characterize the structure of the protein, the effect of the lipid on the protein, and the effect of the protein upon the lipid in its immediate environment. Such techniques can be developed in a reconstituted model system. In such a system it is desirable that the protein adopts the same conformation that it did in the native membrane. Cytochrome b5 is an ideal protein for such a study as it is made on free ribosomes, it is not post-translationally modified and it binds to the membrane via its carboxyl terminus. In addition, the membrane binding domain is relatively small and it contains tryptophans which are fluorescent. The structure of the hydrophobic membrane-binding domain can be investigated by fluorescence quenching by insertion into vesicles made from a series of phospholipids which are brominated at different positions in the acyl chain. The efficiency of quenching gives a measure of the depth, in the membrane, of the fluorescent tryptophans. The quenching in vesicles made from mixtures of brominated and non-brominated lipids indicates the composition of the """"""""boundary lipid"""""""" and the accessibility of the tryptophans to these collisional quenchers. The acyl chains and the polar head groups of the lipids will be systematically varied to determine the role these domains play in determining the protein, and lipid, structure; alternation of the structure of either would modify the quenching. The structure, orientation, and motions of the hydrophobic domain will be influenced by its primary sequence and by the hydrophobic domain. To examine these possibilities protein analogues will be generated by site specific mutagenesis, by chemical synthesis or by enzymic or chemical modification of the cytochrome. CD measurements will give information on secondary structure, NMR on structure and dynamics of proteins and lipids, and calorimetry on lipid structure and the enthalpy of binding. Animal membranes contain over 100 different lipid species. This project will determine the role these lipids play in protein dynamics and topography and the effect of proteins on lipid dynamics and topography.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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University of Virginia
Schools of Medicine
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Ladokhin, A S; Holloway, P W (1995) Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions. Biophys J 69:506-17
Tretyachenko-Ladokhina, V G; Ladokhin, A S; Wang, L et al. (1993) Amino acid substitutions in the membrane-binding domain of cytochrome b5 alter its membrane-binding properties. Biochim Biophys Acta 1153:163-9
Doebler, R; Holloway, P W (1993) A small volume infrared transmission cell for aqueous solutions. Anal Biochem 212:562-4
Ladokhin, A S; Wang, L; Steggles, A W et al. (1993) Fluorescence study of a temperature-induced conversion from the ""loose"" to the ""tight"" binding form of membrane-bound cytochrome b5. Biochemistry 32:6951-6
Ladokhin, A S; Tretyachenko-Ladokhina, V G; Holloway, P W et al. (1992) Biophysical studies of cytochromes B5 with amino acid substitutions in the membrane-binding domain. Biophys J 62:79-81
Ladokhin, A S; Wang, L; Steggles, A W et al. (1991) Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain. Biochemistry 30:10200-6
Bolen, E J; Holloway, P W (1990) Quenching of tryptophan fluorescence by brominated phospholipid. Biochemistry 29:9638-43
Holloway, P W; Buchheit, C (1990) Topography of the membrane-binding domain of cytochrome b5 in lipids by Fourier-transform infrared spectroscopy. Biochemistry 29:9631-7
Holloway, P W; Mantsch, H H (1989) Structure of cytochrome b5 in solution by Fourier-transform infrared spectroscopy. Biochemistry 28:931-5
Holloway, P W; Mantsch, H H (1988) Infrared spectroscopic analysis of salt bridge formation between cytochrome b5 and cytochrome c. Biochemistry 27:7991-3

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