Darrell R. McCaslin

[63] Properties of detergents

Publisher Summary This chapter reviews the use of detergents for solubilization of membranes and as a solvent medium for membrane proteins. It summarizes the properties of detergents that have been used for these purposes and presents some facts relevant to the choice of detergents for particular experiments. The lipids and proteins of a native membrane interact with each other in a complex fashion that differs in detail from membrane to membrane. The optimal detergent for a particular membrane or membrane protein has to be found empirically and may depend on the type of experiment one wants to do. It should be noted that exchange of one detergent for another is relatively simple. One may use different detergents for initial solubilization and delipidation for subsequent protein characterization and for reconstitution into a lipid vesicle. Integrity of a protein may require contacts among polar groups that are outside the lipid bilayer in the native membrane, possibly including interactions with peripheral proteins adjacent to the membrane or with nucleic acid in the case of a nuclear or viral membrane. Such contacts are influenced by pH, ionic strength, and specific ions.

[3] Determination of protein molecular weight in complexes with detergent without knowledge of binding

Publisher Summary This chapter is concerned with the use of sedimentation equilibrium, a procedure that is generally applicable to proteins in the molecular weight range 10,000 to 500,000 and can be used conveniently for protein-amphiphile complexes without interference from non-protein-containing detergent micelles. This chapter presents a summary of the theoretical treatment of sedimentation equilibrium in a centrifugal field for systems containing two or more components and discusses the effects of specific interactions between the protein and solute molecules. It then proceeds to a description of experimental procedures and present examples of the application of this technique to protein-amphiphile complexes. Partial specific volumes are tabulated for amino acid residues, carbohydrates, and a number of detergents and lipids. Atomic and group partial molar volumes that are used to calculate partial specific volumes for chemical compounds are also provided. The chapters consideration is the determination of protein molecular weight in a homogeneous system by a rigorous thermodynamic method.

Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open Escherichia coli RNA Polymerase−λPR Promoter Complexes

Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40-50 bp of DNA immediately upstream of the -35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of ∼13 bp (-11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (-60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAP-λP(R) CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (-100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 ± 0.07) and the OC (0.32 ± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions -100 and +14 on promoter DNA from >300 to <100 Å. The proximity of upstream DNA to the downstream cleft in the advanced CC is consistent with the proposed mechanism for facilitation of OC formation by upstream DNA.

STATE OF AGGREGATION OF MEMBRANE PROTEINS

ABSTRACT Rhodopsin from bovine retinal rods, bacteriorhodopsin from Halobacterium halobium and the calcium-stimulated ATPase from rabbit muscle sarcoplasmic reticulum can be obtained in soluble form in suitable non-denaturing detergents without loss of their native functional or strucutral properties, to the extent that they can be determined. Different detergents, even if closely related, are not equally effective, and results in several detergents have to be compared before one can come to confident conclusions about the state of association in the native membrane. Our studies so far show that bacteriorhodopsin and probably Ca++-ATPase are functionally stable as monomers, which does not imply that they have to be monomers in the native membrane, and bacteriorhodopsin is obviously not, since its native state is crystalline. Rhodopsin is in an oligomeric (probably tetrameric) state in the only detergent studied so far in which bleaching is reversible.