Phylogeny as a guide to structure and function of membrane transport proteins

Protein phylogeny, based on primary amino acid sequence relatedness, reflects the evolutionary process and therefore provides a guide to structure, mechanism and function. Any two proteins that are related by common descent are expected to exhibit similar structures and functions to a degree proportional to the degree of their sequence similarity; but two independently evolving proteins should not. This principle provides the impetus to define protein phylogenetic relationships and interrelate families when possible. In this mini-review, we summarize the computational approaches and criteria we use to establish common evolutionary origin. We apply these tools to define distant superfamily relationships between several previously recognized transport protein families. In some cases, available structural and functional data are evaluated in order to substantiate our claim that molecular phylogeny provides a reliable guide to protein structure and function.     

Oligomeric structure, dynamics, and orientation of membrane proteins from solid-state NMR

Solid-state NMR is a versatile and powerful tool for determining the dynamic structure of membrane proteins at atomic resolution. I review the recent progress in determining the orientation, the internal and global protein dynamics, the oligomeric structure, and the ligand-bound structure of membrane proteins with both alpha-helical and beta sheet conformations. Examples are given that illustrate the insights into protein function that can be gained from the NMR structural information.     

Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni(2+)-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F(0)F(1)-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein.     

Phagosome proteomics: a powerful tool to assess bacteria-mediated immunomodulation

Bacteria-mediated immunomodulation has important implications in microbial infection and bacterial vaccines. Intraphagosomal bacteria negotiate survival niches with the intracellular environment by modulating the phagosome composition during invasion. The final phagosome composition determines the fate of the intraphagosomal bacterium or the efficacy of a bacterial vaccine. Therefore, the phagosome proteome is a valuable readout to assess the ability of a natural or genetically engineered bacterial strain to modulate the host immune response. Compared to a preparation of latex-bead-containing phagosomes, the preparation of bacterial phagosomes requires additional measures to ensure comparable purity due to their closer density to some other organelles. This bottleneck can be overcome with delicate preparation protocols, proper experimental designs to facilitate bioinformatics based discrimination against contaminating proteins, and the incorporation of stable-isotope labeled internal standards to correct for contaminating fractions of phagosomal proteins. The rapid progress in the proteomics and bioinformatics fields provides an array of techniques that promise to bring about an unprecedented coverage of both known and as yet undiscovered immunomodulation pathways within bacterial phagosome proteomes. A precise portrait of the bacteria-mediated immunomodulation pathways in phagosomes will likely aid in the intelligent design of bioengineered bacterial vaccine strains for important future biomedical applications.     

Phylogeny as a guide to structure and function of membrane transport proteins (Review)

Peroxisomes are found in virtually all eukaryotic cells, where they play varied but essential metabolic roles, exemplified by the catastrophic effects of mutations that compromise peroxisome biogenesis and function. This review will aim to provide an accessible introduction to peroxisome biogenesis and protein import for the non-specialist, and draws together recent advances in peroxisome protein targeting and import in plants, animals and yeasts, seeking to define common themes and highlight variations. Despite much progress, many aspects of peroxisome biology remain an enigma, and current questions and controversies in the field are highlighted and discussed.     

Application of a homogenous membrane potential assay to assess mitochondrial function

Decreases in mitochondrial membrane potential (MMP) have been associated with mitochondrial dysfunction that could lead to cell death. The MMP is generated by an electrochemical gradient via the mitochondrial electron transport chain coupled to a series of redox reactions. Measuring the MMP in living cells is commonly used to assess the effect of chemicals on mitochondrial function; decreases in MMP can be detected using lipophilic cationic fluorescent dyes. To identify an optimal dye for use in a high-throughput screening (HTS) format, we compared the ability of mitochondrial membrane potential sensor (Mito-MPS), 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide, rhodamine 123, and tetramethylrhodamine to quantify a decrease in MMP in chemically exposed HepG2 cells cultured in 1,536-well plates. Under the conditions used, the optimal dye for this purpose is Mito-MPS. Next, we developed and optimized a homogenous cell-based Mito-MPS assay for use in 1,536-well plate format and demonstrated the utility of this assay by screening 1,280 compounds in the library of pharmacologically active compounds in HepG2 cells using a quantitative high-throughput screening platform. From the screening, we identified 14 compounds that disrupted the MMP, with half-maximal potencies ranging from 0.15 to 18 μM; among these, compound clusters that contained tyrphostin and 3'-substituted indolone analogs exhibited a structure-activity relationship. Our results demonstrate that this homogenous cell-based Mito-MPS assay can be used to evaluate the ability of large numbers of chemicals to decrease mitochondrial function.     

Hydrogen-deuterium exchange in membrane proteins monitored by IR spectroscopy: a new tool to resolve protein structure and dynamics

As more and more high-resolution structures of proteins become available, the new challenge is the understanding of these small conformational changes that are responsible for protein activity. Specialized difference Fourier transform infrared (FTIR) techniques allow the recording of side-chain modifications or minute secondary structure changes. Yet, large domain movements remain usually unnoticed. FTIR spectroscopy provides a unique opportunity to record (1)H/(2)H exchange kinetics at the level of the amide proton. This approach is extremely sensitive to tertiary structure changes and yields quantitative data on domain/domain interactions. An experimental setup designed for attenuated total reflection and a specific approach for the analysis of the results is described. The study of one membrane protein, the gastric H(+),K(+)-ATPase, demonstrates the usefulness of (1)H/(2)H exchange kinetics for the understanding of the molecular movement related to the catalytic activity.     

The influence of cholesterol on membrane protein structure,function, and dynamics studied by molecular dynamics simulations

The plasma membrane, which encapsulates human cells, is composed of a complex mixture of lipids and embedded proteins. Emerging knowledge points towards the lipids as having a regulating role in protein function. Furthermore, insight from protein crystallography has revealed several different types of lipids intimately bound to membrane proteins and peptides, hereby possibly pointing to a site of action for the observed regulation. Cholesterol is among the lipid membrane constituents most often observed to be co-crystallized with membrane proteins, and the cholesterol levels in cell membranes have been found to play an essential role in health and disease. Remarkably little is known about the mechanism of lipid regulation of membrane protein function in health as well as in disease. Herein, we review molecular dynamics simulation studies aimed at investigating the effect of cholesterol on membrane protein and peptide properties. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Two-dimensional crystals of a membrane protein: arrangement of subunits within the crystal sheet

Two-dimensional crystals have been prepared from the photosynthetic reaction center of Rhodopseudomonas viridis. Filtered images of these crystals show individual subunits approximately 4.5 nm in diameter arranged at a center-to-center distance of 6.4 nm. Our previous studies suggested that each subunit within such a sheet corresponds to a single photosynthetic reaction center. Air-dried and freeze-etched shadowed preparations of the crystals yield images which are quite different from negatively stained material. Rotary-shadowed surfaces of the crystals show rows of wedge-shaped particles separated by 3 nm furrows. Two such wedge-shaped particles occupy the 12.1 X 12.9 nm area in which four negatively stained subunits are normally visualized. Close analysis of these shadowed pictures suggests that both the shadowed and negatively stained images can be accounted for by a single model of subunit arrangement within the crystal. Within each 12.1 X 12.9 nm unit cell, two subunits are placed near one surface of the sheet, and two others are near the other surface. All four subunits are visible in negative stain. When the surface is shadowed, only the two subunits which project above the surface of the sheet accumulate appreciable amounts of the heavy metal shadow. Because of their close position, one subunit shades the other, forming the wedge-shaped appearance characteristic of the crystal. The only arrangement consistent with both shadowed and negatively stained images is one in which the two raised subunits occupy positions at either end of a diagonal across the unit cell. The analysis of shadowed images indicates that the plane group of the crystals is P22(1)2(1).     

Water and bacteriorhodopsin: structure, dynamics, and function

A wealth of information has been gathered during the past decades that water molecules do play an important role in the structure, dynamics, and function of bacteriorhodopsin (bR) and purple membrane. Light-induced structural alterations in bR as detected by X-ray and neutron diffraction at low and high resolution are discussed in relationship to the mechanism of proton pumping. The analysis of high resolution intermediate structures revealed photon-induced rearrangements of water molecules and hydrogen bonds concomitant with conformational changes in the chromophore and the protein. These observations led to an understanding of key features of the pumping mechanism, especially the vectoriality and the different modes of proton translocation in the proton release and uptake domain of bR. In addition, water molecules influence the function of bR via equilibrium fluctuations, which must occur with adequate amplitude so that energy barriers between conformational states can be overcome.     

Topogenesis of membrane proteins: determinants and dynamics

For targeting and integration of proteins into the mammalian endoplasmic reticulum, two types of signals can be distinguished: those that translocate their C-terminal sequence (cleavable signals and signal-anchors) and those that translocate their N-terminus (reverse signal-anchors). In addition to the well established effect of flanking charges, also the length and hydrophobicity of the apolar core of the signal as well as protein folding and glycosylation contribute to orienting the signal in the translocon. In multi-spanning membrane proteins, topogenic determinants are distributed throughout the sequence and may even compete with each other. During topogenesis, segments of up to 60 residues may move back and forth through the translocon, emphasizing unexpected dynamic aspects of topogenesis.     

Two-dimensional separation of erythrocyte membrane proteins.

The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mm EDTA, although dilute NaOH will remove it from the membrane. The Ca²⁺-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Using information theory to assess dynamics, structure, and organization of crayfish agonistic repertoire

Information theory measures provided useful quantitative tools for describing the transfer of information in interacting pairs of the eastern white-river crayfish, Procambarus acutus acutus, and furnished hints for deciphering the dynamics, structure, and organization of the agonistic repertoire in this species. As a confirmation of the predictive and heuristic potentials of the theory, results suggested hypotheses on the mechanisms underlying the formation and maintenance of dominance hierarchies in crayfish and indicated the directions that future developments of the research should follow.     

Two-dimensional crystallization of membrane proteins: the lipid layer strategy

Due to the difficulty to crystallize membrane proteins, there is a considerable interest to intensify research topics aimed at developing new methods of crystallization. In this context, the lipid layer crystallization at the air/water interface, used so far for soluble proteins, has been recently adapted successfully to produce two-dimensional (2D) crystals of membrane proteins, amenable to structural analysis by electron crystallography. Besides to represent a new alternative strategy, this approach gains the advantage to decrease significantly the amount of material needed in incubation trials, thus opening the field of crystallization to those membrane proteins difficult to surexpress and/or purify. The systematic studies that have been performed on different classes of membrane proteins are reviewed and the physico-chemical processes that lead to the production of 2D crystals are addressed. The different drawbacks, advantages and perspectives of this new strategy for providing structural information on membrane proteins are discussed.     

Guanylate cyclase-activating proteins: structure, function, and diversity

The guanylate cyclase-activating proteins (GCAPs), Ca2+-binding proteins of the calmodulin gene superfamily, function as regulators of photoreceptor guanylate cyclases. In contrast to calmodulin, which is active in the Ca2+-bound form, GCAPs stimulate GCs in the [Ca2+]-free form and inhibit GCs upon Ca2+ binding. In vertebrate retinas, at least two GCAP1 and two GCs are present, a third GCAP3 is expressed in humans and fish, and at least five additional GCAP4-8 genes have been identified or are predicted in zebrafish and pufferfish. Missense mutations in GCAP1 (Y99C, I143NT, E155G, and P50L) have been associated with autosomal dominant cone dystrophy. Absence of GCAP1/2 in mice delays recovery of the photoresponse, a phenotype consistent with delay in cGMP synthesis. In the absence of GCAP2, GCAP1 supports the generation of wild-type flash responses in both rod and cone cells. Recent progress revealed an unexpected complexity of the GC-GCAP system, pointing, out a number of unsolved questions.