Tryptophan microstate reshuffling upon the binding of cyclosporin A to human cyclophilin A.
Human cyclophilin A (hCypA) contains one tryptophan residue at position 121 (Trp121). The fluorescence intensity of this single tryptophan residue doubles upon binding the clinically important immunosuppressant cyclosporin A (CsA). Trp121 is in close contact to the bound CsA and is well-conserved in almost all immunophilins. The enhancement of the fluorescence intensity upon binding CsA is investigated by steady-state and time-resolved fluorescence measurements. The crystal structures of hCypA and the complex hCypA-CsA are compared. Only Glu120 is strongly influenced by the binding of CsA. The distance between the indole ring and the carboxylate group doubles during complexation. The influence of Glu120 on the fluorescence properties of Trp121 was investigated by pH-titration, and by substituting glutamate into an aspartate and an alanine residue. The fluorescence measurements on the glutamate mutants reveal that the carboxylate group influences the fluorescence properties of Trp121 to a limited extent. The major effect of CsA binding, however, consists in a reshuffling of the populations of microconformations of Trp121 leading to a selective increase of the 1.5 ns lifetime component. This selection is also accompanied by a decreased polarity of the environment and an increase in the radiative rate constant.
Gastmans
M
M
Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Heverlee, Belgium.
Volckaert
G
G
Engelborghs
Y
Y
D020106:Acrylamide / Q000737:chemistry; D019943:Amino Acid Substitution; D001483:Base Sequence; D016572:Cyclosporine / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D020106:Acrylamide; D016572:Cyclosporine; D014364:Tryptophan; D019696:Peptidylprolyl Isomerase