Dataset Information

Tryptophan rotamer distribution revealed for the ?-helix in tear lipocalin by site-directed tryptophan fluorescence.

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 ?-helical conformations of proteins in solution. Single Trp mutations located in an ?-helical segment of human tear lipocalin were explored for structure assignment. Along with fluorescence ?(max) values, the rotamer model assignment of fluorescence lifetimes fits the backbone conformation. Typically, Trp fluorescence in proteins shows three lifetimes. However, for the ?-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 ?-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 ?-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 ?-helix motif.

SUBMITTER: Gasymov OK

PROVIDER: S-EPMC3517820 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Tryptophan rotamer distribution revealed for the α-helix in tear lipocalin by site-directed tryptophan fluorescence.

Gasymov Oktay K OK Abduragimov Adil R AR Glasgow Ben J BJ

The journal of physical chemistry. B 20121102 45

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 ...[more]

PMID: 23088798

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Project description:In the lipocalin family, the conserved interaction between the main ?-helix and the ?-strand H is an ideal model to study protein side chain dynamics. Site-directed tryptophan fluorescence (SDTF) has successfully elucidated tryptophan rotamers at positions along the main alpha helical segment of tear lipocalin (TL). The rotamers assigned by fluorescent lifetimes of Trp residues corroborate the restriction expected based on secondary structure. Steric conflict constrains Trp residues to two (t, g?) of three possible ?? (t, g?, g?) canonical rotamers. In this study, investigation focused on the interplay between rotamers for a single amino acid position, Trp 130 on the ?-helix and amino acids Val 113 and Leu 115 on the H strand, i.e. long range interactions. Trp130 was substituted for Phe by point mutation (F130W). Mutations at positions 113 and 115 with combinations of Gly, Ala, Phe residues alter the rotamer distribution of Trp130. Mutations, which do not distort local structure, retain two rotamers (two lifetimes) populated in varying proportions. Replacement of either long range partner with a small amino acid, V113A or L115A, eliminates the dominance of the t rotamer. However, a mutation that distorts local structure around Trp130 adds a third fluorescence lifetime component. The results indicate that the energetics of long-range interactions with Trp 130 further tune rotamer populations. Diminished interactions, evident in W130G113A115, result in about a 22% increase of ?-helix content. The data support a hierarchic model of protein folding. Initially the secondary structure is formed by short-range interactions. TL has non-native ?-helix intermediates at this stage. Then, the long-range interactions produce the native fold, in which TL shows ?-helix to ?-sheet transitions. The SDTF method is a valuable tool to assess long-range interaction energies through rotamer distribution as well as the characterization of low-populated rotameric states of functionally important excited protein states.

Project description:Tear lipocalin is a primate protein that was recognized as a lipocalin from the homology of the primary sequence. The protein is most concentrated in tears and produced by lacrimal glands. Tear lipocalin is also produced in the tongue, pituitary, prostate, and the tracheobronchial tree. Tear lipocalin has been assigned a multitude of functions. The functions of tear lipocalin are inexorably linked to structural characteristics that are often shared by the lipocalin family. These characteristics result in the binding and or transport of a wide range of small hydrophobic molecules. The cavity of tear lipocalin is formed by eight strands (A-H) that are arranged in a β-barrel and are joined by loops between the β-strands. Recently, studies of the solution structure of tear lipocalin have unveiled new structural features such as cation-π interactions, which are extant throughout the lipocalin family. Lipocalin has many unique features that affect ligand specificity. These include a capacious and a flexible cavity with mobile and short overhanging loops. Specific features that confer promiscuity for ligand binding in tear lipocalin will be analyzed. The functions of tear lipocalin include the following: antimicrobial activities, scavenger of toxic and tear disruptive compounds, endonuclease activity, and inhibition of cysteine proteases. In addition, tear lipocalin binds and may modulate lipids in the tears. Such actions support roles as an acceptor for phospholipid transfer protein, heteropolymer formation to alter viscosity, and tear surface interactions. The promiscuous lipid-binding properties of tear lipocalin have created opportunities for its use as a drug carrier. Mutant analogs have been created to bind other molecules such as vascular endothelial growth factor for medicinal use. Tear lipocalin has been touted as a useful biomarker for several diseases including breast cancer, chronic obstructive pulmonary disease, diabetic retinopathy, and keratoconus. The functional possibilities of tear lipocalin dramatically expanded when a putative receptor, lipocalin-interacting membrane receptor was identified. However, opposing studies claim that lipocalin-interacting membrane receptor is not specific for lipocalin. A recent study even suggests a different function for the membrane protein. This controversy will be reviewed in light of gene expression data, which suggest that tear lipocalin has a different tissue distribution than the putative receptor. But the data show lipocalin-interacting membrane receptor is expressed on ocular surface epithelium and that a receptor function here would be rational.

Dataset Information

Tryptophan rotamer distribution revealed for the ?-helix in tear lipocalin by site-directed tryptophan fluorescence.

Publications

Tryptophan rotamer distribution revealed for the α-helix in tear lipocalin by site-directed tryptophan fluorescence.

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