title: 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.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Sun
authors.forename: E A
authors.initials: EA
authors.lastname: Pratt
authors.forename: V
authors.initials: V
authors.lastname: Simplaceanu
authors.forename: C
authors.initials: C
authors.lastname: Ho
mesh_terms: D002942:Circular Dichroism; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology*; D005461:Fluorine; D019791:Guanidine; D006146:Guanidines; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D008244:Lysophosphatidylcholines; D009682:Magnetic Resonance Spectroscopy; D008566:Membranes / Q000201:enzymology; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
chemical_list: D003902:Detergents; D006146:Guanidines; D008244:Lysophosphatidylcholines; D012997:Solvents; D005461:Fluorine; D014364:Tryptophan; D007770:L-Lactate Dehydrogenase; D019791:Guanidine