Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

被引:108
作者
Chattopadhyay, K [1 ]
Saffarian, S [1 ]
Elson, EL [1 ]
Frieden, C [1 ]
机构
[1] Washington Univ, Sch Med, Dept Biochem & Mol Biophys, St Louis, MO 63110 USA
关键词
protein folding; conformational dynamics; diffusion coefficient; unfolded state;
D O I
10.1073/pnas.172524899
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Fluorescence correlation spectroscopy (FCS) measurements have been carried out on the intestinal fatty acid binding protein (IFABP) to study microsecond dynamics of the protein in its native state as well as in pH-induced intermediates. IFABP is a small (15 kDa) protein that consists mostly of antiparallel beta-strands enclosing a large central cavity into which the ligand binds. Because this protein does not contain cysteine, two cysteine mutants (Val60Cys and Phe62Cys) have been prepared and covalently modified with fluorescein. Based on fluorescence measurements, one of the mutants (Val60Flu) has the fluorescein moiety inside the cavity of the protein, whereas the fluorescein is exposed to solvent in the other (Phe62Flu). The protein modified at position 60 demonstrates the presence of a conformational event on the order of 35 musec, which is not seen in the other mutant (Phe62Flu). The amplitude of this fast conformational event decreases sharply at low pH as the protein unfolds. Experiments measuring the diffusion as a function of pH indicate the formation of a compact state distinct from the native state at about pH 3.5. Steady state fluorescence and far-UV CD indicates that unfolding occurs at pH values below pH 3.
引用
收藏
页码:14171 / 14176
页数:6
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