Abstract
The use of pressure is an advantageous approach to the study of protein structure and dynamics, because it can shift the equilibrium populations of protein conformations toward higher energy states that are not of sufficient population to be observable at atmospheric pressure. Recently, the Hubbell group at the University of California, Los Angeles, reintroduced the application of high pressure to the study of proteins by electron paramagnetic resonance (EPR) spectroscopy. This methodology is possible using X-band EPR spectroscopy due to advances in pressure intensifiers, sample cells, and resonators. In addition to the commercial availability of the pressure generation and sample cells by Pressure Biosciences Inc., a five-loop–four-gap resonator required for the initial high-pressure EPR spectroscopy experiments by the Hubbell group, and those reported here, was designed by James S. Hyde and built and modified at the National Biomedical EPR Center. With these technological advances, we determined the effect of pressure on the essential periplasmic lipopolysaccharide (LPS) transport protein from Escherichia coli, LptA, and one of its binding partners, LptC. LptA unfolds from the N-terminus to the C-terminus, binding of LPS does not appreciably stabilize the protein under pressure, and monomeric LptA unfolds somewhat more readily than oligomeric LptA upon pressurization to 2 kbar. LptC exhibits a fold and relative lack of stability upon LPS binding similar to LptA, yet adopts an altered, likely monomeric, folded conformation under pressure with only its C-terminus unraveling. The pressure-induced changes likely correlate with functional changes associated with binding and transport of LPS.
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Acknowledgements
The authors thank Jason W. Sidabras and Richard R. Mett for resonator calculations, Matthew Fischer and Kyler Crawford for laboratory assistance, and Jimmy B. Feix for critically reading the manuscript. We also gratefully acknowledge helpful discussions with, and dissemination of the high-pressure EPR methodology by, Wayne L. Hubbell and Michael T. Lerch. Research reported in this publication was supported by the National Institute of Health Grants GM108817 (CSK), RR022422 and OD011937 (DEER instrumentation), and EB001980 (National Biomedical EPR Center at MCW).
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Schultz, K.M., Klug, C.S. High-Pressure EPR Spectroscopy Studies of the E. coli Lipopolysaccharide Transport Proteins LptA and LptC. Appl Magn Reson 48, 1341–1353 (2017). https://doi.org/10.1007/s00723-017-0948-z
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DOI: https://doi.org/10.1007/s00723-017-0948-z