Structure determination of membrane proteins by NMR spectroscopy.

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.     

Structure determination of secondary transport proteins by electron crystallography: two-dimensional crystallization of the betaine uptake system BetP

Structure determination at high resolution is still a challenge for membrane proteins in general, but in particular for secondary transporters due to their highly dynamic nature. X-ray structures of ten secondary transporters have recently been determined, but a thorough understanding of transport mechanisms necessitates structures at different functional states. Electron cryo-microscopy of two-dimensional (2D) crystals offers an alternative to obtain structural information at intermediate resolution. Electron crystallography is a sophisticated way to study proteins in a natural membrane environment and to track conformational changes in situ. Furthermore, basic interactions between protein and lipids can be investigated. Projection and 3-dimensional maps of six secondary transporters from different families have been determined by electron crystallography of 2D crystals at a resolution of 8 A and better. In this review, we give an overview about the principles of 2D crystallization, in particular of secondary transporters, and summarize the important steps successfully applied to establish and improve the 2D crystallization of the high-affinity glycine betaine uptake system from Corynebacterium glutamicum, BetP.     

Extra domains in secondary transport carriers and channel proteins

"Extra" domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA(Fru) and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane alpha-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.     

Extra domains in secondary transport carriers and channel proteins

“Extra” domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA^Fru and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane α-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.     

Mitochondrial structure revealed by high-resolution scanning electron microscopy

Mitochondrial structure has been examined in three dimensions using high-resolution scanning electron microscopy in cells from rat liver, retina (photoreceptors and retinal pigment epithelium), and kidney (proximal convoluted tubular cells and podocytes). Tissues were prepared by aldehyde-osmium fixation and freeze cleavage using a cryoprotectant, followed by removal of the cytosol by immersion in a dilute osmium tetroxide solution. The microscope used (Hitachi S-570) was equipped with a secondary electron detector located in the column above the specimen, situated within the objective lens. Mitochondria in all tissues examined were found to have only tubular cristae, which in some instances could be seen to span the entire diameter of the organelle. The walls of the tubular cristae, when unfractured, were in contact with the inner mitochondrial membrane; and their lumens were open to the intermembranous space. We hypothesize that in cells of many, perhaps most tissues, mitochondrial cristae are not shelf-like but are, in fact, tubes which span the mitochondrial matrix and are continuous with the inner mitochondrial membrane at both ends.     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.     

Yeast kinetochore microtubule dynamics analyzed by high-resolution three-dimensional microscopy

We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.     

High-speed atomic force microscopy: Structure and dynamics of single proteins

For surface analysis of biological molecules, atomic force microscopy (AFM) is an appealing technique combining data acquisition under physiological conditions, for example buffer solution, room temperature and ambient pressure, and high resolution. However, a key feature of life, dynamics, could not be assessed until recently because of the slowness of conventional AFM setups. Thus, for observing bio-molecular processes, the gain of image acquisition speed signifies a key progress. Here, we review the development and recent achievements using high-speed atomic force microscopy (HS-AFM). The HS-AFM is now the only technique to assess structure and dynamics of single molecules, revealing molecular motor action and diffusion dynamics. From this imaging data, watching molecules at work, novel and direct insights could be gained concerning the structure, dynamics and function relationship at the single bio-molecule level.     

Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

The rate at which X-ray structures of membrane proteins are solved is on a par with that of soluble proteins in the late 1970s. There are still many obstacles facing the membrane protein structural community. Recently, there have been several technical achievements in the field that have started to dramatically accelerate structural studies. Here, we summarize these so-called ‘tricks-of-the-trade’ and include case studies of several mammalian transporters.     

Amyloid fibrils of glucagon characterized by high-resolution atomic force microscopy

Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 degrees C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy performed in solution revealed the presence of aggregates after 0.5 h; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity, and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. Atomic force microscopy imaging of pH 2.8 glucagon solutions not agitated or exposed to elevated temperature revealed the presence of amorphous aggregates before the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution Fourier transform infrared spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation were associated with the development of beta-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.     

Hydration in proteins observed by high-resolution neutron crystallography

It is well known that water molecules surrounding a protein play important roles in maintaining its structural stability. Water molecules are known to participate in several physiological processes through the formation of hydrogen bonds. However, the hydration structures of most proteins are not known well at an atomic level at present because X-ray protein crystallography has difficulties to localize hydrogen atoms. In contrast, neutron crystallography has no problem in determining the position of hydrogens with high accuracy.1 In this article, the hydration structures of three proteins are described- myoglobin, wild-type rubredoxin, and a mutant rubredoxin-the structures of which were solved at 1.5- or 1.6-A resolution by neutron structure determination. These hydration patterns show fascinating features and the water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen bond formation and dynamics of hydration. Our results further show that there are strong relationships between these shapes and the water environments.     

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high‐speed super‐resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy‐ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs.