The phenomenon of antimicrobial resistance represents a major public health risk. The activity of integral membrane transporter proteins contributes to antimicrobial resistance in pathogenic bacteria and proton gradient-driven multidrug efflux representatives of the major facilitator superfamily (MFS) of secondary transporters are the dominant antimicrobial efflux proteins in Escherichia coli. In many, but not all, of the characterized MFS multidrug transporters, an aspartic acid residue at position D+5 of the conserved signature Motif A is essential for transport activity. The present work extends those studies to the E. coli MFS multidrug/H+ antiporter MdtM and used a combination of mutagenesis, expression studies, antimicrobial resistance assays, and transport activity measurements to reveal that a negatively charged residue at position D+5 is critical for MdtM transport function.

Recent technical advances have enabled the imaging of single fluorescent molecules. The application of single molecule visualization techniques has opened up new avenues of experimentation in biology at the molecular level. In this article, we review the application of single fluorescent molecule visualization and analysis to an important problem, that of subunit stoichiometry in membrane proteins, with particular emphasis on our approach. Single fluorescent molecules, coupled to fluorescent proteins, are localized in the membranes of cells. The molecules are then exposed to continuous low-level excitation until their fluorescent emissions reach background levels. The high sensitivity of modern instrumentation has enabled direct observations of discrete step decreases in the fluorescence of single molecules, which represent the bleaching of single fluorophores. By counting the number of steps over a large number of single molecules, an average step count is determined from which the stoichiometry is deduced using a binomial model. We examined the stoichiometry of a protein, prestin, that is central to mammalian hearing. We discuss how we prepared, identified, and imaged single molecules of prestin. The methodological considerations behind our approach are described and compared to similar procedures in other laboratories.

To understand how the photoreceptor protein rhodopsin performs in its role as a receptor, its structure needs to be determined at the atomic level. Upon receiving a photon of light, rhodopsin undergoes a change in conformation that allows it to bind and activate the C-protein, transducin. An important future goal should be to determine the structure of both the inactive and the photoactivated state of rhodopsin, R*. This should provide the groundwork necessary for experiments on how rhodopsin achieves its signaling state R*, and how R* functions to activate transducin. To do this, the crystal structure of both rhodopsin and R* must be determined. Few membrane proteins have been successfully crystallized, so this is not a trivial undertaking. Two- or three dimensional crystals of rhodopsin must be prepared that are well ordered, to produce a high-resolution structure. Rhodopsin must be purified to homogeneity and the appropriate detergent(s) selected for crystallization experiments. Long-term thermal stability of the rhodopsin-detergent complex must be achieved in the presence of a precipitant. Two-dimensional crystals may offer advantages in investigating the structure of R*, but the structure obtained may be limited in resolution. It is necessary to work with rhodopsin in the dark, unless suitable light-stable retinal derivatives are developed. Protein engineering of rhodopsin offers attractive opportunities to improve its ability to crystallize, but is presently hindered by the absence of a high-yielding expression system. Knowledge of the structure of rhodopsin will have general importance. Because rhodopsin is a member of the family of C-protein-coupled receptors, knowledge of the structure and the mechanism of action of rhodopsin suggests by analogy how other members of the receptor family may function.

Immunoaffinity chromatography and gel electrophoresis were used to isolate a 44 kDa protein that was bound to a 72 kDa chaperone in Trypanosoma brucei brucei. A polyclonal antiserum to the 44 kDa protein was raised in rats and employed in conjunction with chromatography using DEAE-cellulose, Sephacryl S-300, and hydroxyapatite to purify the protein from membranes of bloodstream forms of the trypanosomes. Immunoblot analysis using this antiserum revealed a protein doublet of 44/45 kDa in T. b. brucei and a single protein band of 53 kDa in almost equivalent amounts throughout the life-cycle stages of T. congolense. Indirect immunofluorescence using affinity-purified antibodies specific for the 44 kDa protein showed labelling of the perinuclear area and reticular system extending throughout the parasites, suggesting that this protein was located in the endoplasmic reticulum. Localization of the 44 kDa molecule in the endoplasmic reticulum was confirmed by immunoelectron microscopy. Protease protection experiments demonstrated that the epitopes bound by antibody were buried within the membrane or towards the lumenal face of the endoplasmic reticulum. Ruthenium Red overlay of nitrocellulose blots containing the 44/45 kDa doublet suggested that the molecules have the potential to bind calcium. The N-terminal amino acid sequence of the 44 kDa protein showed no sequence similarity to any proteins in the database.

Plasmodium falciparum is an intracellular parasite of the red blood cell. During development it exports proteins which are transported to specific locations within the host erythrocyte. We have begun to identify and characterize exported membrane proteins of P.falciparum in order to obtain specific marker molecules for the study of the mechanisms involved in the distribution of parasite-derived proteins within the host cell. In this report we describe the characterization of a 35 kDa protein which is recognized by a monoclonal antibody. The protein is tightly associated with membranes isolated from infected erythrocytes; it is resistant to extraction with alkali and soluble after treatment with detergents. It is located at the membrane of the parasitophorous vacuole and in membrane-bound compartments which appear in the cytoplasm of the infected erythrocyte. The protein co-localizes with the previously described exported protein-1 (exp-1). Considering its localization and physical similarities to exp-1, we name the 35 kDa protein the exported protein-2 (exp-2).

Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a “tripod amphiphile,” that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.

Transmembrane helices are the most readily predictable secondary structure components of proteins. They can be predicted to a high degree of accuracy in a variety of ways. Many of these methods compare new sequence data with the sequence characteristics of known transmembrane domains. However, the known transmembrane sequences are not necessarily representative of a particular organism. We attempt to demonstrate that parameters optimized for the known transmembrane domains are far from optimal when predicting transmembrane regions in a given genome. In particular, we have tested the effect of nucleotide bias upon the composition and hence the prediction characteristics of transmembrane helices. Our analysis shows that nucleotide bias of a genome has a strong and predictable influence upon the occurrences of several of the most important hydrophobic amino acids found within transmembrane helices. Thus, we show that nucleotide bias should be taken into account when determining putative transmembrane domains from sequence data.

Mechanosensation in bacteria involves transducing membrane stress into an electrochemical response. In Escherichia coli and other bacteria, this function is carried out by a number of proteins including MscL, the mechanosensitive channel of large conductance. MscL is the best characterized of all mechanosensitive channels. It has been the subject of numerous structural and functional investigations. The explosion in experimental data on MscL recently culminated in the solution of the three-dimensional structure of the MscL homologue from Mycobacterium tuberculosis. In this review, much of these data are united and interpreted in terms of the newly published M. tuberculosis MscL crystal structure.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the β-barrel of the pore-forming protein α-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that β-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a β-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane β-barrel of α-hemolysin will be very useful in the de novo design of functional membrane proteins based on the β-barrel motif.

To explore the structure of the pore-forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402–424. Each mutant was reacted with a sulfhydryl-specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406–416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402–420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408–R409, is located on the lipid-exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium.