SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii.

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-ß-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleusmarisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers from Multi-Layered or Concentrated Biological Samples.

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

Reconstitution of Detergent-Solubilized Membrane Proteins into Proteoliposomes and Nanodiscs for Functional and Structural Studies.

Reconstitution of detergent-solubilized membrane proteins into phospholipid bilayers allows for functional and structural studies under close-to-native conditions that greatly support protein stability and function. Here we outline the detailed steps for membrane protein reconstitution to result in proteoliposomes and nanodiscs. Reconstitution can be achieved via a number of different strategies. The protocols for preparation of proteoliposomes use detergent removal via dialysis or via nonpolar polystyrene beads, or a mixture of the two methods. In this chapter, the protocols for nanodiscs apply polystyrene beads only. Proteoliposome preparation methods allow for substantial control of the lipid-to-protein ratio, from minimal amounts of phospholipid to high concentrations, type of phospholipid, and mixtures of phospholipids. In addition, dialysis affords a fairly large degree of control and variation of parameters such as rate of reconstitution, temperature, buffer conditions, and proteoliposome size. For the nanodisc approach, which is highly advantageous for ensuring equal access to both membrane sides of the protein as well as fast reconstitution of only a single membrane protein into a well-defined bilayer environment in each nanodisc, the protocols outline how a number of these parameters are more restricted in comparison to the proteoliposome protocols.