NMR and Molecular Genetics as Tools for Investigating Protein Interactions in Membrane Systems

Abstract

AbstractIn our laboratory, we have applied the tools of nuclear magnetic resonance (NMR) spectroscopy and molecular genetics to investigate the structural and dynamic properties of membrane‐associated proteins and their interactions with membrane components. There are two general classes of membrane proteins, i.e., intrinsic and peripheral ones. For the intrinsic membrane proteins, we have chosen the membranebound D‐lactate dehydrogenase (D‐LDH) of Escherichia coli as a model to study protein‐lipid interactions in membranes. D‐LDH is a respiratory enzyme of molecularweight 65, 000 containing flavin adenine dinucleotide (FAD) as a cofactor. The activity of purified D‐LDH is enhanced up to 100‐fold by lipids and detergents. The gene for D‐LDH has been sequenced, and production of the enzyme amplified up to 300‐times normal levels. We have biosynthetically incorporated 5‐fluorotryptophan (5F‐Trp) into D‐LDH and studied the five Trp residues by 19F‐NMR spectroscopy. In order to gain additional information using 19F‐NMR, site‐specific, oligonucleotide‐directed mutagenesis has been used to insert a sixth Trp into D‐LDH at various positions throughout the 571‐amino acid chain. These mutant D‐LDHs are being characterized biochemically and through NMR. For peripheral membrane proteins, we have chosen two periplasmic binding proteins, histidine‐binding protein J (J protein) of Salmonella Typhimurium and glutamine‐binding protein (GlnBP) of E. coli as models to investigate the structure‐function relationship in periplasmic binding protein‐mediated active transport systems. These two proteins both have molecular weights of approximately 25, 000. By using mutant J proteins and GlnBPs and site‐specific, oligonucleotide‐directed mutagenesis techniques, we have assigned several resonances to specific amino acid residues. We are investigating the relationship between ligand‐induced conformational changes in these two proteins and their roles in the active transport of ligand across the cell membrane. We have found that a combination of isotopic labeling, biochemistry, molecular biology, and NMR is a very useful approach to investigate various interactions of membrane‐associated protein systems.