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Title: X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination

Abstract

Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting of individual crystals. In addition, we validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions.

Authors:
 [1];  [1];  [1];  [2];  [1]
  1. Univ. of Illinois at Urbana-Champaign, Urbana, IL (United States)
  2. Argonne National Lab. (ANL), Argonne, IL (United States)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
OSTI Identifier:
1158727
Alternate Identifier(s):
OSTI ID: 1225366
Grant/Contract Number:  
AC02-06CH11357; R01 GM086727
Resource Type:
Journal Article: Published Article
Journal Name:
Crystal Growth and Design
Additional Journal Information:
Journal Volume: 14; Journal Issue: 10; Journal ID: ISSN 1528-7483
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Khvostichenko, Daria S., Schieferstein, Jeremy M., Pawate, Ashtamurthy S., Laible, Philip D., and Kenis, Paul J. A.. X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination. United States: N. p., 2014. Web. doi:10.1021/cg5011488.
Khvostichenko, Daria S., Schieferstein, Jeremy M., Pawate, Ashtamurthy S., Laible, Philip D., & Kenis, Paul J. A.. X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination. United States. doi:10.1021/cg5011488.
Khvostichenko, Daria S., Schieferstein, Jeremy M., Pawate, Ashtamurthy S., Laible, Philip D., and Kenis, Paul J. A.. Thu . "X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination". United States. doi:10.1021/cg5011488.
@article{osti_1158727,
title = {X-ray transparent microfluidic chip for mesophase-based crystallization of membrane proteins and on-chip structure determination},
author = {Khvostichenko, Daria S. and Schieferstein, Jeremy M. and Pawate, Ashtamurthy S. and Laible, Philip D. and Kenis, Paul J. A.},
abstractNote = {Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting of individual crystals. In addition, we validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions.},
doi = {10.1021/cg5011488},
journal = {Crystal Growth and Design},
number = 10,
volume = 14,
place = {United States},
year = {Thu Aug 21 00:00:00 EDT 2014},
month = {Thu Aug 21 00:00:00 EDT 2014}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1021/cg5011488

Citation Metrics:
Cited by: 17 works
Citation information provided by
Web of Science

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