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Circular-dichroism analyses of membrane proteins: examination of environmental effects on bacteriorhodopsin spectra.

ABSTRACT: The secondary structure of bacteriorhodopsin is known from electron-diffraction studies, making bacteriorhodopsin a useful test system for analysing environmental influences on membrane proteins using c.d. spectroscopy. The conformational effects of detergent solubilization and incorporation into vesicles of various types were determined by comparison of the calculated secondary structures derived from c.d. spectra with the structure determined from diffraction studies. In addition, two modified forms of the native purple membrane, a shrunken form of the hexagonal lattice and an orthorhombic lattice form, were used to determine the effects of varying membrane fragment size and protein concentration within the membranes. The two different vesicle incorporation procedures yielded bacteriorhodopsin spectra which were nearly identical with each other and very close to the structure calculated from electron-diffraction studies. Solubilization of the native protein in the non-ionic detergent n-octyl glucoside, without subsequent vesicle incorporation, resulted in a significantly altered protein conformation. Organizing the protein in different membrane lattices produced even more apparent deviations from the secondary structure determined by diffraction studies, as a consequence of optical effects caused by the high protein concentrations in the lattices. These studies show the importance of maintaining a 'native' environment, and the influence of particle geometry in interpreting c.d. studies of membrane proteins.

SUBMITTER: Swords NA

PROVIDER: S-EPMC1132152 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Project description:Cyclic peptides are a promising class of compounds for next-generation antibiotics as they may provide new ways of limiting antibiotic resistance development. Although their cyclic structure will introduce some rigidity, their conformational space is large and they usually have multiple chiral centers that give rise to a wide range of possible stereoisomers. Chiroptical spectroscopies such as vibrational circular dichroism (VCD) are used to assign stereochemistry and discriminate enantiomers of chiral molecules, often in combination with electronic structure methods. The reliable determination of the absolute configuration of cyclic peptides will require robust computational methods than can identify all significant conformers and their relative population and reliably assign their stereochemistry from their chiroptical spectra by comparison with ab initio calculated spectra. We here present a computational protocol for the accurate calculation of the VCD spectra of a series of flexible cyclic oligopeptides. The protocol builds on the Conformer-Rotamer Ensemble Sampling Tool (CREST) developed by Grimme and co-workers ( Phys. Chem. Chem. Phys. 2020, 22, 7169-7192 and J. Chem. Theory. Comput. 2019, 15, 2847-2862) in combination with postoptimizations using B3LYP and moderately sized basis sets. Our recommended computational protocol for the computation of VCD spectra of cyclic oligopeptides consists of three steps: (1) conformational sampling with CREST and tight-binding density functional theory (xTB); (2) energy ranking based on single-point energy calculations as well as geometry optimization and VCD calculations of conformers that are within 2.5 kcal/mol of the most stable conformer using B3LYP/6-31+G*/CPCM; and (3) VCD spectra generation based on Boltzmann weighting with Gibbs free energies. Our protocol provides a feasible basis for generating VCD spectra also for larger cyclic peptides of biological/pharmaceutical interest and can thus be used to investigate promising compounds for next-generation antibiotics.

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Circular-dichroism analyses of membrane proteins: examination of environmental effects on bacteriorhodopsin spectra.

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