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Electron spin-echo envelope modulation (ESEEM) reveals water and phosphate interactions with the KcsA potassium channel.

ABSTRACT: Electron spin-echo envelope modulation (ESEEM) spectroscopy is a well-established technique for the study of naturally occurring paramagnetic metal centers. The technique has been used to study copper complexes, hemes, enzyme mechanisms, micellar water content, and water permeation profiles in membranes, among other applications. In the present study, we combine ESEEM spectroscopy with site-directed spin labeling (SDSL) and X-ray crystallography in order to evaluate the technique's potential as a structural tool to describe the native environment of membrane proteins. Using the KcsA potassium channel as a model system, we demonstrate that deuterium ESEEM can detect water permeation along the lipid-exposed surface of the KcsA outer helix. We further demonstrate that (31)P ESEEM is able to identify channel residues that interact with the phosphate headgroup of the lipid bilayer. In combination with X-ray crystallography, the (31)P data may be used to define the phosphate interaction surface of the protein. The results presented here establish ESEEM as a highly informative technique for SDSL studies of membrane proteins.

SUBMITTER: Cieslak JA

PROVIDER: S-EPMC2840637 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Electron spin-echo envelope modulation (ESEEM) reveals water and phosphate interactions with the KcsA potassium channel.

Cieslak John A JA Focia Pamela J PJ Gross Adrian A

Biochemistry 20100201 7

Electron spin-echo envelope modulation (ESEEM) spectroscopy is a well-established technique for the study of naturally occurring paramagnetic metal centers. The technique has been used to study copper complexes, hemes, enzyme mechanisms, micellar water content, and water permeation profiles in membranes, among other applications. In the present study, we combine ESEEM spectroscopy with site-directed spin labeling (SDSL) and X-ray crystallography in order to evaluate the technique's potential as ...[more]

PMID: 20092291

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Project description:This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of electron spin echo envelope modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an α-helical secondary structure with ESEEM spectroscopy using a (2)H-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins. (2)H-SL distance distributions from the MD results indicated that (2)H-labeled Leu was a very strong candidate to significantly improve this ESEEM approach. In order to test this hypothesis, the secondary structure of the α-helical M2δ peptide of the acetylcholine receptor (AChR) incorporated into a bicelle was investigated with (2)H-labeled Leu d(10) at position 10 (i) and nitroxide spin labels positioned 1, 2, 3, and 4 residues away (denoted i+1 to i+4) with ESEEM spectroscopy. The ESEEM data reveal a unique pattern that is characteristic of an α-helix (3.6 residues per turn). Strong (2)H modulation was detected for the i+3 and i+4 samples, but not for the i+2 sample. The (2)H modulation depth observed for (2)H-labeled d(10) Leu was significantly enhanced (×4) when compared to previous ESEEM measurements that used (2)H-labeled d(8) Val. Computational studies indicate that deuterium nuclei on the Leu side chain are closer to the spin label when compared to Val. The enhancement of (2)H modulation and the corresponding Fourier Transform (FT) peak intensity for (2)H-labeled Leu significantly reduces the ESEEM data acquisition time for Leu when compared to Val. This research demonstrates that a different (2)H-labeled amino acid residue can be used as an efficient ESEEM probe further substantiating this important biophysical technique. Finally, this new method can provide pertinent qualitative structural information on membrane proteins in a short time (few minutes) at low sample concentrations (~50 μM).

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Electron spin-echo envelope modulation (ESEEM) reveals water and phosphate interactions with the KcsA potassium channel.

Publications

Electron spin-echo envelope modulation (ESEEM) reveals water and phosphate interactions with the KcsA potassium channel.

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