Conformational states of the switch I region of Ha-ras-p21 in hinge residue mutants studied by fluorescence lifetime and fluorescence anisotropy measurements.
The hinge residues (Val29 and Ile36) of the switch I region (also known as the effector loop) of the Ha-ras-p21 protein have been mutated to glycines to accelerate the conformational changes typical for the effector loop. In this work, we have studied the influence of the combined mutations on the steady-state structure of the switch I region of the protein in both the inactive GDP-bound conformation as in the active GTP-bound conformation. Here, we use the fluorescence properties of the single tryptophan residue in the Y32W mutant of Ha-ras-p21. This mutant has already been used extensively as a reference form of the protein. Reducing the size of the side chains of the hinge residues not only accelerates the conformational changes but also affects the steady-state structures of the effector loop as indicated by the changes in the fluorescence properties. A thorough analysis of the fluorescence changes (quantum yield, lifetimes, etc.) proves that these changes are from a reshuffling between the rotamer populations of Trp. The population reshuffling is caused by the overall structural rearrangement along the switch I region. The effects are clearly more pronounced in the inactive GDP-bound conformation than in the active GTP-bound conformation. The effect of both mutations seems to be additive in the GDP-bound state, but cooperative in the GTP-bound state.
Kuppens
Steven
S
Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium.
Hellings
Mario
M
Jordens
Jan
J
Verheyden
Stefan
S
Engelborghs
Yves
Y
D019943:Amino Acid Substitution; D005453:Fluorescence; D005454:Fluorescence Polarization; D005470:Fluorometry; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D006207:Half-Life; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry
D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)