Investigation of the four cooperative unfolding units existing in the MICAL-1 CH domain.
The solution structure of human MICAL-1 calpolnin homology (CH) domain is composed of six alpha helices and one 3(10) helix. To study the unfolding of this domain, we carry out native-state hydrogen exchange, intrinsic fluorescence and far-UV circular dichroism experiments. The free energy of unfolding, DeltaG(H2O), is calculated to be 7.11+/-0.58 kcal mol(-1) from GuHCl denaturation at pH 6.5. Four cooperative unfolding units are found using native-state hydrogen exchange experiment. Forty-seven slow-exchange residues can be studied by native-state hydrogen exchange experiments. From the concentration dependence of exchange rates, free energy of amide hydrogen with solvent, DeltaG(HX) and m-value (sensitivity of exposure to denaturant) are obtained, which reveal four cooperative unfolding units. The slowest exchanging protons are distributed throughout the whole hydrophobic core of the protein, which might be the folding core. These results will help us understand the structure of MICAL-1 CH domain more deeply.
Hefei National Laboratory for Physical Sciences at Microscale, and School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China.
Xianju
X
Jin
Jiahai
J
Zhang
Haiming
H
Dai
Hongbin
H
Sun
Dandan
D
Wang
Jihui
J
Wu
Yunyu
Y
Shi
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002942:Circular Dichroism; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D060588:LIM Domain Proteins; D008840:Microfilament Proteins / Q000737:chemistry; D006899:Mixed Function Oxygenases; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D000096985:Calponins
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D019791:Guanidine; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D006899:Mixed Function Oxygenases; D012997:Solvents; D000096985:Calponins; C455272:MICAL1 protein, human