Reversible Conjugation of Non-ionic Detergent Micelles Promotes Partitioning of Membrane Proteins under Non-denaturing Conditions.

Mitra Lal,Guy Patchornik,Mordechai Sheves,Ellen Wachtel

doi:10.1021/acs.langmuir.1c03343

Abstract

In the decades’-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein aggregation in aqueous solution and/or as a conformation stabilizing environment. We have focused on exploiting the physical chemistry of detergent micelles in order to direct intrinsic MP/detergent complexes to assemble via conjugation under ambient conditions, thereby permitting finely tuned control over the micelle cloud point. In the current work, three commercially available amphiphilic, bipyridine chelators in combination with Fe2+ or Ni2+ were tested for their ability to conjugate non-ionic detergent micelles both in the presence and absence of an encapsulated bacteriorhodopsin molecule. Water-soluble chelators were added, and results were monitored with light microscopy and dynamic light scattering (DLS). [Bipyridine:metal] complexes produced micellar conjugates, which appeared as oil-rich globules (10–200 μm) under a light microscope. DLS analysis demonstrated that micellar conjugation is complete 20 min after the introduction of the amphiphilic complex, and that the conjugation process can be fully or partially reversed with water-soluble chelators. This process of controlled conjugation/deconjugation under nondenaturing conditions provides broader flexibility in the choice of detergent for intrinsic MP purification and conformational flexibility during the crystallization procedure.

Highlights

Centrifugation at a relative centrifugal force (RCF) 21,000; and 400 μL of the resulting supernatant was used for determining the hydrodynamic size distribution. (II) Conjugated micelles: Micellar conjugation was initiated by adding 24 μL of 15 mM DP-nonyl and
Following 20 min incubation at 19 °C, dynamic light scattering (DLS) measurements were rings in the bipyridine moiety is bound to an aliphatic tail comprising nine carbons (Figure 1B), thereby anchoring the immediately performed. (III) Process reversibility with ethylenediaminetetraacetic acid (EDTA) or chelator in the micelle core
We found that overnight incubation at 19 °C of bR protein detergent complexes (PDCs) with the three amphiphilic complexes led to purple globules (Figure 5A−C)

Summary

Introduction

Detergent micelles are dynamic structures.[1−5] Whereas dilute aqueous micellar suspensions may be regarded as ideal solutions in which micelles do not interact,[6] physical and/or chemical changes in the micellar environment can promote their interactions: (a) inclusion of polymeric precipitants; (b) increase in ionic strength; or (c) change in temperature.[1,6] Initially isotropic and transparent solutions become turbid, that is, the cloud point is reached, beyond which phase separation into a detergent-rich phase and a detergent-poor phase occurs.[1,6,7] Such phase separation has been exploited for intrinsic membrane protein (MP) purification.[8−10] MPs partition spontaneously into the detergent-rich phase, whereas more hydrophilic proteins are rejected.[8,9] partial purification of MPs from water-soluble and/or more polar proteins is achieved, and the contaminating background present in MP preparations can be removed. Protein purification relies on detergents capable of reaching cloud point conditions at temperatures that preserve the native conformation of the target MP. To date, this has been achieved primarily with the non-ionic surfactant Triton X-114 (cloud point 22−23 °C).[8−10] Similar considerations direct MP crystallization protocols.[11]

Methods

Results

Conclusion