A 19F-NMR study of the equilibrium unfolding of membrane-associated D-lactate dehydrogenase of Escherichia coli.
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.
Sun
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ZY
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Pratt
E A
EA
Simplaceanu
V
V
Ho
C
C
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
D003902:Detergents; D006146:Guanidines; D008244:Lysophosphatidylcholines; D012997:Solvents; D005461:Fluorine; D014364:Tryptophan; D007770:L-Lactate Dehydrogenase; D019791:Guanidine