Strategy for membrane protein crystallization exemplified with OmpA and OmpX.
ABSTRACT The bacterial outer membrane proteins OmpA and OmpX were modified in such a manner that they yielded bulky crystals diffracting X-rays isotropically beyond 2 A resolution and permitting detailed structural analyses. The procedure involved semi-directed mutagenesis, mass production into inclusion bodies, and (re)naturation therefrom; it should be applicable for a broader range of membrane proteins.
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ABSTRACT: Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8-35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting 'OligAPol' have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8-35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications.Nucleic Acids Research 04/2014; · 8.81 Impact Factor
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ABSTRACT: Thirty-three years have elapsed since the first membrane protein (MP) was brought back in vitro to its native state starting from the completely unfolded polypeptide. Folding MPs is as useful from a practical point of view as it is thought-provoking from a theoretical one. Yet, this activity is considered as a high-risk, time-consuming endeavor, full of pitfalls, its path littered with the broken careers of graduate students sacrificed on the altar of a long shot that never paid off. In fact, a surprisingly high number of MPs have actually been folded or refolded in vitro. Analysis of the literature indicates i)that the endeavor is not as desperate as it may seem, ii)that techniques are diversifying and improving, and iii)that many MPs do not need the cellular biosynthetic apparatus, nor even a membrane environment, to reach a functional 3D structure. A compilation, hopefully close to complete, is presented of MPs that have been (re)folded in vitro to-date, with the conditions of their synthesis, the denaturant(s) used, if any, and the (re)folding conditions, along with a few comments. The hope is that this analysis will encourage membrane protein biochemists to consider producing their target proteins in this way, help them decide about an experimental course, and stimulate the reflection about which environments favor membrane protein folding and why.Archives of Biochemistry and Biophysics 07/2014; · 3.37 Impact Factor
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ABSTRACT: Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.Journal of Membrane Biology 03/2014; · 2.48 Impact Factor