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Toward structure determination using membrane-protein nanocrystals and microcrystals.

ABSTRACT: Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only 100 unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet brought fully-hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses (?70fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. During the initial experiments at the AMO beamline using 6.9-? wavelength, Bragg peaks were recorded to 8.5-? resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage [94]. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.

SUBMITTER: Hunter MS

PROVIDER: S-EPMC3414265 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Toward structure determination using membrane-protein nanocrystals and microcrystals.

Hunter Mark S MS Fromme Petra P

Methods (San Diego, Calif.) 20111222 4

Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high- ...[more]

PMID: 22197730

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Project description:The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane ?-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three-dimensional structure and the orientation of the protein within the membrane. When combined with dihedral angle restraints derived from analysis of isotropic chemical shifts, molecular fragment replacement, and de novo structure prediction, orientation restraints can yield high-quality three-dimensional structures with few or no distance restraints. Using complementary solid-state NMR methods based on oriented sample (OS) and magic angle spinning (MAS) approaches, one can resolve and assign multiple peaks through the use of (15)N/(13)C labeled samples and measure precise restraints to determine structures.

Project description:Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20?µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2A adenosine receptor (A2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8?000?000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05?Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20?µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

Project description:The crystal structure of the β(2)-adrenergic receptor in complex with an agonist and its cognate G protein has just recently been determined. It is now possible to explore in molecular detail the means by which this paradigmatic transmembrane receptor binds agonist, communicates the impulse or signaling event across the membrane, and sets in motion a series of G protein-directed intracellular responses. The structure was determined using crystals of the ternary complex grown in a rationally designed lipidic mesophase by the so-called in meso method. The method is proving to be particularly useful in the G protein-coupled receptor field where the structures of 13 distinct receptor types have been determined in the past 5 years. In addition to receptors, the method has proven to be useful with a wide variety of integral membrane protein classes that include bacterial and eukaryotic rhodopsins, light-harvesting complex II (LHII), photosynthetic reaction centers, cytochrome oxidases, β-barrels, an exchanger, and an integral membrane peptide. This attests to the versatility and range of the method and supports the view that the in meso method should be included in the arsenal of the serious membrane structural biologist. For this to happen, however, the reluctance to adopt it attributable, in part, to the anticipated difficulties associated with handling the sticky, viscous cubic mesophase in which crystals grow must be overcome. Harvesting and collecting diffraction data with the mesophase-grown crystals are also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. Over the years, we have endeavored to establish how the method works at a molecular level and to make it user-friendly. To these ends, tools for handling the mesophase in the pico- to nanoliter volume range have been developed for highly efficient crystallization screening in manual and robotic modes. Methods have been implemented for evaluating the functional activity of membrane proteins reconstituted into the bilayer of the cubic phase as a prelude to crystallogenesis. Glass crystallization plates that provide unparalleled optical quality and sensitivity to nascent crystals have been built. Lipid and precipitant screens have been designed for a more rational approach to crystallogenesis such that the method can now be applied to an even wider variety of membrane protein types. In this work, these assorted advances are outlined along with a summary of the membrane proteins that have yielded to the method. The prospects for and the challenges that must be overcome to further develop the method are described.

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Toward structure determination using membrane-protein nanocrystals and microcrystals.

Publications

Toward structure determination using membrane-protein nanocrystals and microcrystals.

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