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Published October 29, 2008 | Accepted Version + Supplemental Material
Journal Article Open

Simple Host-Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

Abstract

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

Additional Information

© 2008 American Chemical Society. Published In Issue: October 29, 2008. Article ASAP: October 03, 2008. Received: July 10, 2008. This work was supported in part through the NIH Roadmap for Medical Research (R01 GM075827-01) and UC/ANL Collaborative Seed Funding. We thank ATCG3D funded by the NIGMS and NCRR under the PSI-2 Specialized Center program (U54 GM074961) for providing some of the equipment used in this work. V.T. was supported by ATCG3D. A.M.S. was supported by an EPSRC LSI Postdoctoral Fellowship. S.N. was supported by the NIH Roadmap Physical and Chemical Biology undergraduate training program at UC. We would like to thank Eva Chi for discussion about DLS experiments and Elizabeth W. Boyd for contributions in writing and editing this manuscript. Use of the Argonne National Laboratory LS-CAT beamlines, BioCARS beamlines, and GM/CA beamlines at the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. DE-AC02-06CH11357. GM/CA CAT has been funded in whole or in part with Federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Science (Y1-GM-1104). Use of the BioCARS Sector 14 was supported by the National Institutes of Health, National Center for Research Resources, under Grant Number RR07707. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (Grant 085P1000817). Supporting Information: Details of chemicals and equipment used, experimental details for the preparation of samples for DLS and TLC, the details of the Labview program used to control the microfluidic crystallization method, and results obtained for control experiments and the construction of calibration curves. This material is available free of charge via the Internet at http://pubs.acs.org/.

Attached Files

Accepted Version - nihms-79622.pdf

Supplemental Material - ismagilov_detergent_capture_JACS_130_2008_14324_LL_SI.pdf

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August 19, 2023
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