A ¹⁹F-NMR Study of the Equilibrium Unfolding of Membrane-Associated d-Lactate Dehydrogenase of Escherichia coli

Abstract

Partially folded protein intermediates have been observed by 19F-NMR spectroscopy during the equilibrium unfolding of the membrane-associated D-lactate dehydrogenase (D-LDH) of Escherichia coli by a denaturant, guanidine hydrochloride (Gdn.HCl). The results from 19F-NMR and circular dichroism spectroscopic studies suggest that the intermediates observed at low Gdn.HCl concentrations (< 3.5 M) exhibit features similar to "molten globules" that contain considerable amounts of secondary and tertiary structure. The results of 19F-NMR studies on 5F-Trp-labeled D-LDH, such as the chemical shift changes, nuclear Overhauser effect, and solvent-induced isotopic shift effect, show that different regions of D-LDH unfold nonuniformly in Gdn.HCl in the presence of lysophosphatidylcholine. The polypeptide appears to unfold in a general order from the carboxyl end to the amino end, in agreement with previous findings from our laboratory that the carboxyl-terminal region of D-LDH is largely exposed to the solvent while the amino-terminal region is buried in the protein core. The structure of the partially unfolded intermediate forms of D-LDH is stabilized in the presence of lipid-like detergents, such as lysophosphatidylcholine.

The crystal structure of d- lactate dehydrogenase, a peripheral membrane respiratory enzyme

“…This complementary electrostatic interaction presumably would stabilize the orientation of the protein on the membrane surface. Our model for protein-membrane association is supported by previous studies showing that D-LDH in the presence of detergents containing charged head groups, such as hexadecyltrimethylammonium bromide and lysophosphatidylcholine, has a better defined structure than in the presence of nonionic detergent (octyl glucoside) (36). In fact, other peripheral membrane proteins are known to contain clusters of basic residues that facilitate membrane adsorption (2,3).…”

Fluorine as an NMR probe for structural studies of chemical and biological systems

“…Another fluoro derivative of tryptophan, 6-F-Trp, was used in a study of the equilibrium unfolding of membraneassociated D-lactate dehydrogenase (D-LDH) of E. coli, a 65 kDa protein and one of the primary dehydrogenases in the bacterial respiratory chain. 55 The stability of different regions in D-LDH and the interaction of partially unfolded protein intermediates ('molten globules') with detergent micelles were investigated.…”

Fluorine-19 NMR of integral membrane proteins illustrated with studies of GPCRs

“…19 F-NMR labels incorporated into a protein can provide information on the local environment of the labels, including solvent exposure, and on the conformational states and dynamics of the protein [3,4,5,6 •• ]. 19 F-NMR experiments with integral membrane proteins further enable characterization of the variations of motional and structural properties in different environments, such as lipid vesicles, organic solvents and detergents [7,8,9,10,11,12,13,14,15,16,17,18 •• ,19 • ,20 • ,21 • ]. …”