Conformational dynamics of the GdmHCl-induced molten globule state of creatine kinase monitored by hydrogen exchange and mass spectrometry.
Our understanding of the mechanism of protein folding can be improved by the characterization of folding intermediate states. Intrinsic tryptophan fluorescence measurements of equilibrium GdmHCl-induced unfolding of MM-CK allow for the construction of a "phase diagram", which shows the presence of five different conformational states, including three partially folded intermediates. However, only three states are detected by using pulsed-labeled H-D exchange analyzed by electrospray ionization mass spectrometry. One of them is the native state, and the two other species are present in proportions strongly dependent on the GdmHCl concentration and denaturation time. The low-mass peak is due to a largely exchange-incompetent state, which has gained only approximately 10 deuteriums more than the native protein. This population of MM-CK molecules has undergone a small conformational change induced by low GdmHCl concentrations. However, this limited change is in itself not sufficient to inactivate the enzyme or is easily reversible. The high-mass peak corresponds to a population of MM-CK that is fully deuterated. The comparison of fluorescence, activity, and H-D exchange measurements shows that the maximally populated intermediate at 0.8 M GdmHCl has the characteristics of a molten globule. It has no activity; it has 55% of its native alpha-helices and a maximum fluorescence emission wavelength of approximately 341 nm, and it binds ANS strongly. However, no protection against exchange is detected under the conditions used in this work. This paradox, the presence of significant residual secondary and tertiary structures detected by optical probes and the total deuteration of its amide protons detected by H-D exchange and mass spectrometry, could be explained by a highly dynamic MM-CK molten globule.
Mazon
Hortense
H
UMR CNRS 5013, Biomembranes et enzymes associés, Université Claude Bernard Lyon I, 43, boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
Marcillat
Olivier
O
Forest
Eric
E
Smith
David L
DL
Vial
Christian
C
D000818:Animals; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry; D019791:Guanidine / Q000494:pharmacology; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature
D006859:Hydrogen; D003903:Deuterium; D003402:Creatine Kinase; D019791:Guanidine