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Title: Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods

Abstract

Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of 'ordered' waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamicsmore » is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
DOE - OFFICE OF SCIENCE
OSTI Identifier:
1019860
Report Number(s):
BNL-95706-2011-JA
Journal ID: ISSN 0006-2960; TRN: US201115%%496
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 49; Journal Issue: 5
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; CATALYSIS; CELL CONSTITUENTS; CHAINS; CHEMICAL REACTIONS; COMPARTMENTS; CONFORMATIONAL CHANGES; DETECTION; FUNCTIONALS; MASS SPECTROSCOPY; MEMBRANE PROTEINS; PROTEIN STRUCTURE; PROTEINS; RADIOLYSIS; RESIDUES; STABILIZATION; WATER; X-RAY DIFFRACTION; national synchrotron light source

Citation Formats

Orban, T., Gupta, S, Palczewski, K, and Chance, M. Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods. United States: N. p., 2010. Web. doi:10.1021/bi901889t.
Orban, T., Gupta, S, Palczewski, K, & Chance, M. Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods. United States. doi:10.1021/bi901889t.
Orban, T., Gupta, S, Palczewski, K, and Chance, M. Fri . "Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods". United States. doi:10.1021/bi901889t.
@article{osti_1019860,
title = {Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods},
author = {Orban, T. and Gupta, S and Palczewski, K and Chance, M},
abstractNote = {Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of 'ordered' waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.},
doi = {10.1021/bi901889t},
journal = {Biochemistry},
number = 5,
volume = 49,
place = {United States},
year = {Fri Jan 01 00:00:00 EST 2010},
month = {Fri Jan 01 00:00:00 EST 2010}
}
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  • We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and environmental electron receptors. LC/MS/MS analysis resulted in the identification of about 79% membrane proteins among all proteins identified from the enriched sample. To illustrate the quantification of membrane proteome changes, enriched membrane protein samplesmore » from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) were further labeled with 16O and 18O at the peptide level prior to LC-MS analysis. A chemical-probe-labeled pure protein has also been used as an internal standard for normalization purpose. The quantitative data revealed reduced abundances of many outer membrane proteins such as OmcA and MtrC in ΔgspD mutant cells, which agreed well with previously published studies.« less