Mapping membrane protein structure with fluorescence

Membrane proteins regulate many cellular processes including signaling cascades, ion transport, membrane fusion, and cell-to-cell communications. Understanding the architecture and conformational fluctuations of these proteins is critical to understanding their regulation and functions. Fluorescence methods including intensity mapping, fluorescence resonance energy transfer (FRET), and photo-induced electron transfer, allow for targeted measurements of domains within membrane proteins. These methods can reveal how a protein is structured and how it transitions between different conformational states. Here, I will review recent work done using fluorescence to map the structures of membrane proteins, focusing on how each of these methods can be applied to understanding the dynamic nature of individual membrane proteins and protein complexes.     

Understanding membrane protein structure by design

In contrast to soluble proteins, the primary interactions that specify and stabilize membrane protein structures are still largely a matter of speculation. Although van der Waals interactions have been gaining increasing favor as the dominant player, new results demonstrate the strength of hydrogen bonding in a membrane environment.     

Overcoming barriers to membrane protein structure determination

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.     

Comparative analysis of membrane protein structure databases

BackgroundMembrane proteins play important roles in cell survival and cell communication, as they function as transporters, receptors, anchors and enzymes. They are also potential targets for drugs that block receptors or inhibit enzymes related to diseases. Although the number of known structures of membrane proteins is still small relative to the size of the proteome as a whole, many new membrane protein structures have been determined recently.Scope of the articleWe compared and analyzed the widely used membrane protein databases, mpstruc, Orientations of Proteins in Membranes (OPM), and PDBTM, as well as the extended dataset of mpstruc based on sequence similarity, the PDB structures whose classification field indicates that they are “membrane proteins” and the proteins with Structural Classification of Proteins (SCOP) class-f domains. We evaluated the relationships between these databases or datasets based on the overlap in their contents and the degree of consistency in the structural, topological, and functional classifications and in the transmembrane domain assignment.Major conclusionsThe membrane databases differ from each other in their coverage, and in the criteria that they use for annotation and classification. To ensure the efficient use of these databases, it is important to understand their differences and similarities. The establishment of more detailed and consistent annotations for the sequence, structure, membrane association, and function of membrane proteins is still required.General significanceConsidering the recent growth of experimentally determined structures, a broad survey and cumulative analysis of the sum of knowledge as presented in the membrane protein structure databases can be helpful to elucidate structures and functions of membrane proteins. We also aim to provide a framework for future research and classification of membrane proteins.     

Current status of membrane protein structure classification

For over 2 decades, continuous efforts to organize the jungle of available protein structures have been underway. Although a number of discrepancies between different classification approaches for soluble proteins have been reported, the classification of membrane proteins has so far not been comparatively studied because of the limited amount of available structural data. Here, we present an analysis of α‐helical membrane protein classification in the SCOP and CATH databases. In the current set of 63 α‐helical membrane protein chains having between 1 and 13 transmembrane helices, we observed a number of differently classified proteins both regarding their domain and fold assignment. The majority of all discrepancies affect single transmembrane helix, two helix hairpin, and four helix bundle domains, while domains with more than five helices are mostly classified consistently between SCOP and CATH. It thus appears that the structural constraints imposed by the lipid bilayer complicate the classification of membrane proteins with only few membrane‐spanning regions. This problem seems to be specific for membrane proteins as soluble four helix bundles, not restrained by the membrane, are more consistently classified by SCOP and CATH. Our findings indicate that the structural space of small membrane helix bundles is highly continuous such that even minor differences in individual classification procedures may lead to a significantly different classification. Membrane proteins with few helices and limited structural diversity only seem to be reasonably classifiable if the definition of a fold is adapted to include more fine‐grained structural features such as helix–helix interactions and reentrant regions. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The progress of membrane protein structure determination

The rate of membrane protein (MP) structure determination has been examined for the 18-year period following the publication of the first high-resolution crystal structure. The growth is solidly exponential, but lags behind the rate for soluble proteins during the equivalent time period.     

The secretory carrier membrane protein family: structure and membrane topology

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.     

Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.     

Solution structure of peptides from HIV-1 Vpr protein that cause membrane permeabilization and growth arrest

Vpr, one of the accessory gene products encoded by HIV-1, is a 96-residue protein with a number of functions, including targeting of the viral pre-integration complex to the nucleus and inducing growth arrest of dividing cells. We have characterized by 2D NMR the solution conformations of bioactive synthetic peptide fragments of Vpr encompassing a pair of H(F/S)RIG sequence motifs (residues 71–75 and 78–82 of HIV-1 Vpr) that cause cell membrane permeabilization and death in yeast and mammalian cells. Due to limited solubility of the peptides in water, their structures were studied in aqueous trifluoroethanol. Peptide Vpr^59–86 (residues 59–86 of Vpr) formed an α-helix encompassing residues 60–77, with a kink in the vicinity of residue 62. The first of the repeated sequence motifs (HFRIG) participated in the well-defined α-helical domain whereas the second (HSRIG) lay outside the helical domain and formed a reverse turn followed by a less ordered region. On the other hand, peptides Vpr^71–82 and Vpr^71–96, in which the sequence motifs were located at the N-terminus, were largely unstructured under similar conditions, as judged by their C^αH chemical shifts. Thus, the HFRIG and HSRIG motifs adopt α-helical and turn structures, respectively, when preceded by a helical structure, but are largely unstructured in isolation. The implications of these findings for interpretation of the structure–function relationships of synthetic peptides containing these motifs are discussed. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Microscopic assessment of membrane protein structure and function

Membrane proteins represent an important class of proteins that are encoded by about 40% of all genes, but compared to soluble proteins structural information is sparse. Most of the atomic coordinates currently available are from bacterial membrane proteins and have been obtained by X-ray crystallography. Recent results demonstrate the imaging power of the atomic force microscope and the accuracy of electron crystallography. These methods allow membrane proteins to be studied while embedded in the bilayer, and thus in a functional state. The low signal-to-noise ratio of cryoelectron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review discusses examples of microscopic membrane protein structure determination and illuminates recent progress.     

Scoring function accuracy for membrane protein structure prediction

We perform a systematic examination of the ability of several different high-resolution, atomic-detail scoring functions to discriminate native conformations of loops in membrane proteins from non-native but physically reasonable, or "decoy," conformations. Decoys constructed from changing a loop conformation while keeping the remainder of the protein fixed are a challenging test of energy function accuracy. Nevertheless, the best of the energy functions we examined recognized the native structure as lowest in energy around half the time, and consistently chose it as a low-energy structure. This suggests that the best of present energy functions, even without a representation of the lipid bilayer, are of sufficient accuracy to give reasonable confidence in predictions of membrane protein structure. We also constructed homology models for each structure, using other known structures in the same protein family as templates. Homology models were constructed using several scoring functions and modeling programs, but with a comparable sampling effort for each procedure. Our results indicate that the quality of sequence alignment is probably the most important factor in model accuracy for sequence identity from 20-40%; one can expect a reasonably accurate model for membrane proteins when sequence identity is greater than 30%, in agreement with previous studies. Most errors are localized in loop regions, which tend to be found outside the lipid bilayer. For the most discriminative energy functions, it appears that errors are most likely due to lack of sufficient sampling, although it should be stressed that present energy functions are still far from perfectly reliable.     

NMR structure of the integral membrane protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.     

Molecular dynamics simulations and membrane protein structure quality

Despite a growing repertoire of membrane protein structures (currently ∼120 unique structures), considerations of low resolution and crystallization in the absence of a lipid bilayer require the development of techniques to assess the global quality of membrane protein folds. This is also the case for assessment of, e.g. homology models of human membrane proteins based on structures of (distant) bacterial homologues. Molecular dynamics (MD) simulations may be used to help evaluate the quality of a membrane protein structure or model. We have used a structure of the bacterial ABC transporter MsbA which has the correct transmembrane helices but an incorrect handedness and topology of their packing to test simulation methods of quality assessment. An MD simulation of the MsbA model in a lipid bilayer is compared to a simulation of another bacterial ABC transporter, BtuCD. The latter structure has demonstrated good conformational stability in the same bilayer environment and over the same timescale (20 ns) as for the MsbA model simulation. A number of comparative analyses of the two simulations were performed to assess changes in the structural integrity of each protein. The results show a significant difference between the two simulations, chiefly due to the dramatic structural deformations of MsbA. We therefore propose that MD could become a useful quality control tool for membrane protein structural biology. In particular, it provides a way in which to explore the global conformational stability of a model membrane protein fold.     

Side-chain contributions to membrane protein structure and stability

The molecular forces that stabilize membrane protein structure are poorly understood. To investigate these forces we introduced alanine substitutions at 24 positions in the B helix of bacteriorhodopsin and examined their effects on structure and stability. Although most of the results can be rationalized in terms of the folded structure, there are a number of surprises. (1) We find a remarkably high frequency of stabilizing mutations (17%), indicating that membrane proteins are not highly optimized for stability. (2) Helix B is kinked, with the kink centered around Pro50. The P50A mutation has no effect on stability, however, and a crystal structure reveals that the helix remains bent, indicating that tertiary contacts dominate in the distortion of this helix. (3) We find that the protein is stabilized by about 1kcal/mol for every 38A(2) of surface area buried, which is quite similar to soluble proteins in spite of their dramatically different environments. (4) We find little energetic difference, on average, in the burial of apolar surface or polar surface area, implying that van der Waals packing is the dominant force that drives membrane protein folding.     

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.     

Progress in the analysis of membrane protein structure and function

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.     

膜蛋白结构研究方法新进展

膜蛋白是一类结构独特的蛋白质,执行很多基本的和重要的细胞生物学功能。了解膜蛋白在生物膜上的基本构象,对研究膜蛋白的精细拓扑结构、功能具有重要意义。但是膜蛋白的疏水特性使其需要与生物膜共同形成稳定的自然构象,至今在蛋白质组学的研究中对膜蛋白知之甚少。了解分子结构是了解生物大分子功能的一个重要途径。因此,本文对近来膜分子结构研究领域的进展作一简要概述。晶体学方法、单颗粒方法和原子力显微镜为膜蛋白的研究提供了大量的细节数据。固相核磁共振技术提供了跨膜α螺旋结构的方向约束数据和精确的分子间距离约束数据。直接位点标记的旋转电子顺磁共振可以得到更长的距离约束数据,但是目前的标记策略仍然具有局限性。位点特异的红外二色性分析可使得在脂双层中定向分析跨膜α螺旋束成为可能。     

The Plasmodium rhoptry associated protein complex is important for parasitophorous vacuole membrane structure and intraerythrocytic parasite growth

Plasmodium parasites must invade erythrocytes in order to cause the disease malaria. The invasion process involves the coordinated secretion of parasite proteins from apical organelles that include the rhoptries. The rhoptry is comprised of two compartments: the neck and the bulb. Rhoptry neck proteins are involved in host cell adhesion and formation of the tight junction that forms between the invading parasite and erythrocyte, whereas the role of rhoptry bulb proteins remains ill‐defined due to the lack of functional studies. In this study, we show that the rhoptry‐associated protein (RAP) complex is not required for rhoptry morphology or erythrocyte invasion. Instead, post‐invasion when the parasite is bounded by a parasitophorous vacuolar membrane (PVM), the RAP complex facilitates the survival of the parasite in its new intracellular environment. Consequently, conditional knockdown of members of the RAP complex leads to altered PVM structure, delayed intra‐erythrocytic growth, and reduced parasitaemias in infected mice. This study provides evidence that rhoptry bulb proteins localising to the parasite–host cell interface are not simply by‐products of the invasion process but contribute to the growth of Plasmodium in vivo.