Abstract
Rotamer libraries are a valuable tool for protein structure determination, modeling, and design. Site-directed tryptophan fluorescence (SDTF) was used in combination with the rotamer model for the fluorescence intensity decays to solve α-helical conformations of proteins in solution. Single Trp mutations located in an α-helical segment of human tear lipocalin were explored for structure assignment. Along with fluorescence λ(max) values, the rotamer model assignment of fluorescence lifetimes fits the backbone conformation. Typically, Trp fluorescence in proteins shows three lifetimes. However, for the α-helix, two lifetimes assigned to t and g(-) rotamers were satisfactory to describe Trp fluorescence intensity decays. The g(+) rotamer is not feasible in the α-helix due to steric restriction. Trp rotamer distributions obtained by fluorescence were compared with the rotamer library derived from X-ray crystallography data of proteins. The Trp rotamer distributions vary for solvent exposed and buried (tertiary interaction) sites. A new strategy using the rotamer distribution with SDTF (RD-SDTF) removes the limitation of regular SDTF and other labeling techniques, in which site-specific differences, e.g., accessibility, are presumed. The RD-SDTF technique does not rely on environmental differences of side chains and is able to detect α-helical structure where all side chains are exposed to solvent. Potentially, this technique is applicable to various proteins including membrane proteins, which are rich in α-helix motif.