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 indicates 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 MmIAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP to 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.

2D Electron Crystallography of Membrane Protein Single-, Double-, and Multi-Layered Ordered Arrays.

The electron cryo-microscopy (cryo-EM) approach of 2D electron crystallography allows for structure determination of two-dimensional (2D) crystals of soluble and membrane proteins, employing identical principles and methods once 2D crystals are obtained. Two-dimensional crystallization trials of membrane proteins can result in multiple outcomes of ordered arrays, which may be suited for either 2D electron crystallography, helical analysis, or MicroED.The membrane protein 2D crystals used for 2D electron crystallography are either single- or double-layered ordered proteoliposome vesicles or sheet-like membranes. We have developed a cryo-EM grid preparation approach, which allows for the analysis of stacked 2D crystals that are neither suitable for MicroED nor for directly applying 2D electron crystallography. This new grid preparation approach, the peel-blot, uses the capillary force generated by submicron filter paper and mechanical means for the separation of stacked 2D crystals into single-layered 2D crystals, for which standard 2D electron crystallography can then be employed. The preparation of 2D crystals, the peel-blot grid preparation, and the structure determination by 2D electron crystallography are described here.

ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure-function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 µs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.

Complementary Mutations in the N and L Proteins for Restoration of Viral RNA Synthesis.

During viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif-Glu169, Phe171, and Leu174-to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid.IMPORTANCE During viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis.

Inducing two-dimensional crystallization of membrane proteins by dialysis for electron crystallography.

Electron crystallography is an electron cryo-microscopy (cryo-EM) method that is particularly suitable for structure-function studies of small membrane proteins, which are crystallized in two-dimensional (2D) arrays for subsequent cryo-EM data collection and image processing. This approach allows for structural analysis of membrane proteins in a close-to-native, phospholipid bilayer environment. The process of growing 2D crystals from purified membrane proteins by dialysis detergent removal is described in this chapter. A short section covers screening for and identifying 2D crystals by transmission electron microscopy, and in the last section, optimization of the purification to obtain crystals of higher quality is discussed.

Two-dimensional crystallization by dialysis for structural studies of membrane proteins by the cryo-EM method electron crystallography.

Two-dimensional (2D) crystals of integral membrane proteins, comprising ordered protein reconstituted into a synthetic lipid bilayer, can be induced to form from detergent solubilized and purified membrane protein sources via the addition of exogenous lipid and the subsequent removal of the solubilizing detergent. This is most commonly accomplished by dialysis of a small volume of ternary protein-detergent-lipid mixture against a large volume of buffer, and can be carried out using common, easily available materials. Following successful crystallization, electron crystallographic data obtained by electron cryo-microscopy (cryo-EM) of vitrified 2D crystals can be used to determine the structure of the lipid bilayer-embedded integral membrane protein.

Two-dimensional crystallization of membrane proteins by reconstitution through dialysis.

Studies of membrane proteins by two-dimensional (2D) crystallization and electron crystallography have provided crucial information on the structure and function of a rapidly growing number of these intricate proteins within a close-to-native lipid bilayer. Here we provide protocols for planning and executing 2D crystallization trials by detergent removal through dialysis, including the preparation of phospholipids and the dialysis setup. General factors to be considered, such as the protein preparation, solubilizing detergent, lipid for reconstitution, and buffer conditions are discussed. Several 2D crystallization conditions are highlighted that have shown great promise to grow 2D crystals within a surprisingly short amount of time. Finally, conditions for optimizing order and size of 2D crystals are outlined.

Screening for two-dimensional crystals by transmission electron microscopy of negatively stained samples.

Structural studies of soluble and membrane proteins by electron crystallography include several critical steps. While the two-dimensional (2D) crystallization arguably may be described as the major bottleneck of electron crystallography, the screening by transmission electron microscopy (EM) to identify 2D crystals requires great care as well as practice. Both sample preparation and EM are skills that are relatively easily acquired, compared to the identification of the first ordered arrays. Added to this, membranes may have a variety of morphologies and sizes. Here we describe all steps involved in the screening for 2D crystals as well as the evaluation of samples.

Structure-function insights of membrane and soluble proteins revealed by electron crystallography.

Electron crystallography is emerging as an important method in solving protein structures. While it has found extensive applications in the understanding of membrane protein structure and function at a wide range of resolutions, from revealing oligomeric arrangements to atomic models, electron crystallography has also provided invaluable information on the soluble α/ß-tubulin which could not be obtained by any other method to date. Examples of critical insights from selected structures of membrane proteins as well as α/ß-tubulin are described here, demonstrating the vast potential of electron crystallography that is first beginning to unfold.

Kinetically controlled overgrowth of Ag or Au on Pd nanocrystal seeds: from hybrid dimers to nonconcentric and concentric bimetallic nanocrystals.

This article describes a systematic study of the nucleation and growth of Ag (and Au) on Pd nanocrystal seeds. By carefully controlling the reaction kinetics, the newly formed Ag atoms could be directed to selectively nucleate and then epitaxially grow on a specific number (ranging from one to six) of the six faces on a cubic Pd seed, leading to the formation of bimetallic nanocrystals with a variety of different structures. In addition to changing the injection rate of precursor, we also systematically investigated other reaction parameters including the capping agent, reductant, and reaction temperature. Our results suggest that the site-selective growth of Ag on cubic Pd seeds could be readily realized by optimizing these reaction parameters. On the basis of the positions of Pd seeds inside the bimetallic nanocrystals as revealed by TEM imaging and elemental mapping, we could identify the exact growth pathways and achieve a clear and thorough understanding of the mechanisms. We have successfully applied the same strategy based on kinetic control to cubic Pd seeds with different sizes and octahedral Pd seeds of one size to generate an array of novel bimetallic nanocrystals with well-controlled structures. With cubic Pd seeds as an example, we have also extended this strategy to the Pd-Au system. We believe this work will provide a promising route to the fabrication of bimetallic nanocrystals with novel structures and properties for applications in plasmonics, catalysis, and other areas.

Aggregation and interaction of cationic nanoparticles on bacterial surfaces.

Cationic monolayer-protected gold nanoparticles (AuNPs) with sizes of 6 or 2 nm interact with the cell membranes of Escherichia coli (Gram-) and Bacillus subtilis (Gram+), resulting in the formation of strikingly distinct AuNP surface aggregation patterns or lysis depending upon the size of the AuNPs. The aggregation phenomena were investigated by transmission electron microscopy and UV-vis spectroscopy. Upon proteolytic treatment of the bacteria, the distinct aggregation patterns disappeared.

Amyloid fibril formation by the glaucoma-associated olfactomedin domain of myocilin.

Myocilin is a protein found in the extracellular matrix of trabecular meshwork tissue, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset inherited glaucoma. Elevated levels and aggregation of myocilin hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of reports on the intracellular accumulation of mutant and WT myocilin in vitro, cell culture, and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation-dependent and self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting.

Cryo-EM in the study of membrane transport proteins.

Electron cryomicroscopy (cryo-EM) has evolved as a widely used approach to understand a range of structure-function questions, particularly of membrane proteins. Studies by both electron crystallography and single particle analysis have provided a wealth of information on membrane transport proteins. Cryo-EM methods with an emphasis on electron crystallography, which has yielded the most membrane transport protein structural information of any of the cryo-EM techniques, are described here. Two-dimensional crystallization approaches are outlined, as well as advances in cryo-EM specimen preparation, data collection, and image processing. Examples of membrane transport protein structure described serve to illustrate some of the advances in both structural understanding and methods. Further examples outline impressive results that were obtained by a combination of electron crystallography and X-ray crystallography as well as additional complementary methods.

Electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.

Membrane protein structure and function can be studied by two powerful and highly complementary electron cryomicroscopy (cryo-EM) methods: electron crystallography of two-dimensional (2D) crystals and single particle analysis of detergent-solubilized protein complexes. To obtain the highest-possible resolution data from membrane proteins, whether prepared as 2D crystals or single particles, cryo-EM samples must be vitrified with great care. Grid preparation for cryo-EM of 2D crystals is possible by back-injection, the carbon sandwich technique, drying in sugars before cooling in the electron microscope, or plunge-freezing. Specimen grids for single particle cryo-EM studies of membrane proteins are usually produced by plunge-freezing protein solutions, supported either by perforated or a continuous carbon film substrate. This review outlines the different techniques available and the suitability of each method for particular samples and studies. Experimental considerations in sample preparation and preservation include the protein itself and the presence of lipid or detergent. The appearance of cryo-EM samples in different conditions is also discussed.

Assessing two-dimensional crystallization trials of small membrane proteins for structural biology studies by electron crystallography.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions. By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size. While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Electron crystallography of membrane proteins: two-dimensional crystallization and screening by electron microscopy.

Structural and functional information of membrane proteins at ever-increasing resolution is being obtained by electron crystallography. While a large amount of work on the development of methods for electron microscopy and image processing has resulted in tremendous advances in terms of speed of data collection and resolution, general guidelines for crystallization are first starting to emerge. Yet two-dimensional crystallization itself will always remain the limiting factor of this powerful approach in structural biology. Two-dimensional crystallization through detergent removal by dialysis is the most widely used technique. Four main factors need to be considered for the dialysis method: the protein preparation, the detergent, the lipid added as well as any constituent lipid, and the buffer conditions. Equally important is proper and careful screening to identify two-dimensional crystals.

Two-dimensional crystallization of human vitamin K-dependent gamma-glutamyl carboxylase.

Planar-tubular two-dimensional (2D) crystals of human vitamin K-dependent gamma-glutamyl carboxylase grow in the presence of dimyristoyl phosphatidylcholine (DMPC). Surprisingly, these crystals form below the phase transition temperature of DMPC and at the unusually low molar lipid-to-protein (LPR) ratio of 1, while 2D crystals are conventionally grown above the phase transition temperature of the reconstituting lipid and significantly higher LPRs. The crystals are up to 0.75 microm in the shorter dimension of the planar tubes and at least 1 microm in length. Due to the planar-tubular nature of the crystals, two lattices are present. These are rotated by nearly 90 degrees in respect to each other. The ordered arrays exhibit p12(1) plane group symmetry with unit cell dimensions of a=83.7 A, b=76.6 A, gamma=91 degrees. Projection maps calculated from images of negatively stained and electron cryo-microscopy samples reveal the human vitamin K-dependent gamma-glutamyl carboxylase to be a monomer.