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Site-directed fluorescence approaches to monitor the structural dynamics of proteins using intrinsic Trp and labeled with extrinsic fluorophores.

ABSTRACT: Comprehensive understanding of a protein's function depends on having reliable, sophisticated tools to study protein structural dynamics in physiologically-relevant conditions. Here, we present an effective, robust step-by-step protocol to monitor the structural dynamics (including hydration dynamics) of a protein utilizing various site-directed fluorescence (SDFL) approaches. This protocol should be widely applicable for studying soluble proteins, intrinsically-disordered proteins, and membrane proteins. For complete details on the use and execution of this protocol, please refer to Das et al. (2020), Das and Raghuraman (2021), and Chatterjee et al. (2021).

SUBMITTER: Brahma R

PROVIDER: S-EPMC8889417 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Site-directed fluorescence approaches to monitor the structural dynamics of proteins using intrinsic Trp and labeled with extrinsic fluorophores.

Brahma Rupasree R Das Anindita A Raghuraman H H

STAR protocols 20220228 1

Comprehensive understanding of a protein's function depends on having reliable, sophisticated tools to study protein structural dynamics in physiologically-relevant conditions. Here, we present an effective, robust step-by-step protocol to monitor the structural dynamics (including hydration dynamics) of a protein utilizing various site-directed fluorescence (SDFL) approaches. This protocol should be widely applicable for studying soluble proteins, intrinsically-disordered proteins, and membrane ...[more]

PMID: 35252885

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Project description: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.

Dataset Information

Site-directed fluorescence approaches to monitor the structural dynamics of proteins using intrinsic Trp and labeled with extrinsic fluorophores.

Publications

Site-directed fluorescence approaches to monitor the structural dynamics of proteins using intrinsic Trp and labeled with extrinsic fluorophores.

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