Abstract
In the lipocalin family, the conserved interaction between the main α-helix and the β-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 χ1 (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 α-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 α-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 α-helix intermediates at this stage. Then, the long-range interactions produce the native fold, in which TL shows α-helix to β-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.
Highlights
Molecular interactions with proteins are regulated through conformational changes that are hierarchical in time and space [1,2,3]
The rotameric distribution model derived from site-directed tryptophan fluorescence (SDTF) (RD-SDTF) uses a three-site jump rotamer model of x1 (180u (t); 260u (g2); +60u (g+)) to assign Trp fluorescence lifetimes [13,22]
Changes observed in the CD spectrum of W130 compared to that of native tear lipocalin (TL) (Fig. 2B) have been attributed to alteration in packing of secondary structural elements generated by the introduction of a side-chain bulkier than Phe130 (Fig. 1B) [37]
Summary
Molecular interactions with proteins are regulated through conformational changes that are hierarchical in time and space [1,2,3]. Side-chain rotamers distributions and\or redistributions observed in the conformational transitions are pivotal mechanistic features of protein functions. Side-chain rotamer libraries have been widely used in theoretical conformational and X-ray crystallographic models. Site-directed tryptophan fluorescence (SDTF) was used to assign rotameric distributions in the alpha helix of tear lipocalin and to detect conformational changes involving side-chain rearrangement [13]. The rotameric distribution model derived from SDTF (RD-SDTF) uses a three-site jump rotamer model of x1 (180u (t); 260u (g2); +60u (g+)) to assign Trp fluorescence lifetimes [13,22]. Rotamer libraries derived from the extensive X-ray crystallographic data support a model with three canonical rotamers for x1 angles [23,24,25]. Non-canonical rotamers (x1 angles) comprise less than 1% of the rotamer library
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