Modeling membrane protein structure through site-directed ESR spectroscopy

Site-directed spin labeling (SDSL) electron spin resonance (ESR) spectroscopy is a relatively new biophysical tool for obtaining structural information about proteins. This thesis presents a novel approach, based on powerful spectral analysis techniques (multicomponent spectral simulations and evolutionary optimizations of ESR spectra) and modeling of the protein structure by calculating the restrictions of the conformational space of the attached spin label. First, the feasibility of the ESR spectral analysis was enhanced by speeding-up the spectrum optimization and by automation of the analysis routines to enable the handling of large sets of spectroscopic data (e.g., for the joint analysis of SDSL-ESR spectra from multiple sites of a spin-labeled protein). According to the testing examples a speed-up factor of 5-7 was achieved. Secondly, SDSL-ESR was used to study the topology of the long N-terminal domain of the photosynthetic light-harvesting complex CP29. Wild-type protein containing a single cysteine at position 108 and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases the apoproteins were either solubilized in detergent, or they were reconstituted with their native pigments in vitro. The spin label ESR spectra were analyzed in terms of a multi-component spectral simulation approach. These results permit to trace the structural organization of the long Nterminal domain of CP29 leading to a structural model for its N-terminal domain. Thirdly, we proposed a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. The proposed structural model takes into account the restricting effect of the protein backbone, amino acid side chains and lipid environment. We tested the sensitivity of this approach for artificial oligopeptides and then for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels by employing highthroughput site-directed mutagenesis. We found a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to an α-helix. Finally, by using an optimization algorithm we optimized the parameters of the protein-lipid model by improving the fit of the simulation data to the experimental conformational space data. The outcome of the optimization was a family of best-fit structures of membrane-embedded M13 protein, which not only agree with the available SDSL-ESR data, but also was consistent with a recent model based on site-directed fluorescence labeling. Therefore, the present method provides a challenging starting point for the development of a powerful methodology for the protein structure characterization, an alternative approach to conventional techniques.

Bibliographic Details

Main Author:	Kavalenka, A.A.
Other Authors:	van Amerongen, Herbert
Format:	Doctoral thesis biblioteca
Language:	English
Subjects:	electron paramagnetic resonance spectroscopy, molecular conformation, spectroscopy, surface proteins, moleculaire structuur, oppervlakte-eiwitten, paramagnetische elektronenresonantiespectroscopie, spectroscopie,
Online Access:	https://research.wur.nl/en/publications/modeling-membrane-protein-structure-through-site-directed-esr-spe
Tags:	Add Tag No Tags, Be the first to tag this record!