A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Newby, Zachary E.; O’Connell, Joseph D.; Gruswitz, Franz; Hays, Franklin A.; Harries, William; Harwood, Ian M; Ho, Joseph; Lee, John K.; Savage, David F.; Miercke, Larry J. W.; Stroud, Robert M.

doi:10.1038/nprot.2009.27

Cited by 108 publications

(89 citation statements)

References 83 publications

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“…BOG has been widely used as a mild lipid-mimicking detergent to induce and stabilize amphipathic -helical structure in solution and during protein crystallization (48). The detergent has been used in numerous studies to investigate the effects of lipid environment in the helical WT apoA-I (45).…”

Section: Interaction With Bogmentioning

confidence: 99%

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Mei

Liu

Herscovitz

et al. 2016

Journal of Lipid Research

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Section: Interaction With Bogmentioning

confidence: 99%

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Mei

Liu

Herscovitz

et al. 2016

Journal of Lipid Research

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“…S7). ELIC overexpression, purification, crystallization, and structure determination were performed essentially as described by Hilf and Dutzler (9) with minor modifications (39). Because of the limited resolution of the X-ray data, no attempt was made to force the side chains in our models to adopt conformations that correspond to rotamers or that maximize the number of hydrogen bonds formed.…”

Section: Methodsmentioning

confidence: 99%

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

González-Gutiérrez

Lukk

Agarwal

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinicreceptor superfamily.electrophysiology | structure-function T he use of bacterial and archaeal ion channels as models of the structure and function of their eukaryotic counterparts has become one of the mainstays of ion-channel biophysics. It seems to us that this practice is particularly well justified whenever the use of noneukaryotic channels facilitates experiments that are otherwise too difficult or too cumbersome to perform. A clear case in point is the determination of structural models of these membrane proteins using X-ray crystallography.One of the main goals of direct structural approaches as applied to ion channels is to determine what these proteins look like in their different functional states. Assuming that all of these conformations can form well-diffracting crystals, the problem is reduced to finding the conditions under which the occupancy of each particular state is maximized. In general, for well-characterized ion channels, the experimental maneuvers that are needed to favor or disfavor at least some of these different conformations are known. For example, in the case of (wild-type) neurotransmitter-gated channels, the binding of the natural neurotransmitter strongly stabilizes the desensitized state (e.g., ref. 1), whereas the absence of neurotransmitter or the binding of a competitive antagonist keeps the channel mostly closed (2, 3). Similarly, mut...

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“…Although, in general, the detergent used for solubilization does not need to be the same as the one used for purification and crystallization, it is advisable to ensure maximum homogeneity of the final sample [29]. To produce homogeneous and soluble HtpX, OG was used to extract the protein initially from the membranes at 2% and thereafter maintained at 1%.…”

Section: Large-scale Production Of Homogeneous Htpxmentioning

confidence: 99%

Expression and purification of integral membrane metallopeptidase HtpX

Arolas

García‐Castellanos

Goulas

et al. 2014

Protein Expression and Purification

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The authors state they have no competing financial interest Arolas et al. ! 2! Highlights• The integral membrane metallopeptidase HtpX was overexpressed in E. coli.• A catalytically ablated HtpX mutant was designed to avoid self-cleavage.• The protein was extracted from membranes and purified in octyl-!-D-glucoside.• The protein was purified by cobalt-affinity, anion-exchange and size-exclusion.• The overall system renders milligram amounts of HtpX and fosters structural studies. AbstractLittle is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli.Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His 8 -tag, extracted from the membranes using octyl-!-D-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and sizeexclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members.

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A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Cited by 108 publications

References 83 publications

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Probing the C-terminal domain of lipid-free apoA-I demonstrates the vital role of the H10B sequence repeat in HDL formation

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

Expression and purification of integral membrane metallopeptidase HtpX

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