Dirk-Jan Slotboom

A structural classification of substrate-binding proteins.

Substrate‐binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP‐binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA‐binding proteins, as well as channels and receptors from both pro‐ and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three‐dimensional structure.

Optimization of membrane protein overexpression and purification using GFP fusions

Optimizing conditions for the overexpression and purification of membrane proteins for functional and structural studies is usually a laborious and time-consuming process. This process can be accelerated using membrane protein–GFP fusions 1–3 , which allows direct monitoring and visualization of membrane proteins of interest at any stage during overexpression, solubilization and purification (Fig. 1). The exceptionally stable GFP moiety of the fusion protein can be used to detect membrane proteins by observing fluorescence in whole cells during overexpression, with a detection limit as low as 10 µg of GFP per liter of culture, and in solution during solubilization and purification. Notably, the fluorescence of the GFP moiety can also be detected in standard SDS polyacrylamide gels with a detection limit of less than 5 ng of GFP per protein band (Fig. 2). In-gel fluorescence allows assessment of the integrity of membrane protein–GFP fusions and provides a rapid and generic alternative for the notoriously difficult immunoblotting of membrane proteins. With whole-cell and in-gel fluorescence the overexpression potential of many membrane protein–GFP fusions can be rapidly assessed and yields of promising targets can be improved. In this protocol the Escherichia coli BL21(DE3)-pET system—the most widely used (membrane) protein overexpression system—is used as a platform to illustrate the GFP-based method. The methodology described in this protocol can be transferred easily to other systems.

Lactococcus lactis as host for overproduction of functional membrane proteins

Lactococcus lactis has many properties that are ideal for enhanced expression of membrane proteins. The organism is easy and inexpensive to culture, has a single membrane and relatively mild proteolytic activity. Methods for genetic manipulation are fully established and a tightly controlled promoter system is available, with which the level of expression can be varied with the inducer concentration. Here we describe our experiences with lactococcal expression of the mechanosensitive channel, the human KDEL receptor and transporters belonging to the ABC transporter family, the major facilitator superfamily, the mitochondrial carrier family and the peptide transporter family. Previously published expression studies only deal with the overexpression of prokaryotic membrane proteins, but in this paper, experimental data are presented for the overproduction of mitochondrial and hydrogenosomal carriers and the human KDEL receptor. These eukaryotic membrane proteins were expressed in a functional form and at levels amenable to structural work.

Quality control of overexpressed membrane proteins

Overexpression of membrane proteins in Escherichia coli frequently leads to the formation of aggregates or inclusion bodies, which is undesirable for most studies. Ideally, one would like to optimize the expression conditions by monitoring simultaneously and rapidly both the amounts of properly folded and aggregated membrane protein, a requirement not met by any of the currently available methods. Here, we describe a simple gel-based approach with green fluorescent protein as folding indicator to detect well folded and aggregated proteins simultaneously. The method allows for rapid screening and, importantly, pinpointing the most likely bottlenecks in protein production.

A scalable, GFP-based pipeline for membrane protein overexpression screening and purification

We describe a generic, GFP‐based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane‐integral flavocytochrome. The approach is scalable and suitable for high‐throughput applications. The GFP‐based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.

Na+ : Aspartate Coupling Stoichiometry in the Glutamate Transporter Homologue Glt(Ph)

The Na(+) aspartate symporter Glt(Ph) from Pyrococcus horikoshii is the only member of the glutamate transporter family for which crystal structures have been determined. The cation:aspartate coupling stoichiometry is unknown, thus hampering the elucidation of the ion coupling mechanism. Here we measure transport of (22)Na(+) and [(14)C]aspartate in proteoliposomes containing purified Glt(Ph) and demonstrate that three Na(+) ions are symported with aspartate.

A Novel ADP/ATP Transporter in the Mitosome of the Microaerophilic Human Parasite Entamoeba histolytica

Recent data suggest that microaerophilic and parasitic protozoa, which lack oxidative phosphorylation, nevertheless contain mitochondrial homologs [1-6], organelles that share common ancestry with mitochondria. Such widespread retention suggests there may be a common function for mitochondrial homologs that makes them essential for eukaryotic cells. We determined the mitochondrial carrier family (MCF) complement of the Entamoeba histolytica mitochondrial homolog, also known as a crypton [5] or more commonly as a mitosome [3]. MCF proteins support mitochondrial metabolic energy generation, DNA replication, and amino-acid metabolism by linking biochemical pathways in the mitochondrial matrix with those in the cytosol [7]. MCF diversity thus closely mirrors important facets of mitochondrial metabolic diversity. The Entamoeba histolytica mitosome has lost all but a single type of MCF protein, which transports ATP and ADP via a novel mechanism that is not reliant on a membrane potential. Phylogenetic analyses confirm that the Entamoeba ADP/ATP carrier is distinct from archetypal mitochondrial ADP/ATP carriers, an observation that is supported by its different substrate and inhibitor specificity. Because many functions of yeast and human mitochondria rely on solutes transported by specialized members of this family, the Entamoeba mitosome must contain only a small subset of these processes requiring adenine nucleotide exchange.

Functional expression of eukaryotic membrane proteins in Lactococcus lactis

The overproduction of eukaryotic membrane proteins is a major impediment in their structural and functional characterization. Here we have used the nisin‐inducible expression system of Lactococcus lactis for the overproduction of 11 mitochondrial transport proteins from yeast. They were expressed at high levels in a functional state in the cytoplasmic membrane. The results also show that the level of expression is influenced by the N‐terminal regions of the transporters. Expression levels were improved >10‐fold either by replacing or truncating these regions or by adding lactococcal signal peptides. The observed expression levels are now compatible with a realistic exploration of crystallization conditions. The lactococcal expression system may be used for the high‐throughput functional characterization of eukaryotic membrane proteins and structural genomics.

The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes

Mitochondrial carriers are believed widely to be dimers both in structure and function. However, the structural fold is a barrel of six transmembrane α-helices without an obvious dimerisation interface. Here, we show by negative dominance studies that the yeast mitochondrial ADP/ATP carrier 2 from Saccharomyces cerevisiae (AAC2) is functional as a monomer in the mitochondrial membrane. Adenine nucleotide transport by wild-type AAC2 is inhibited by the sulfhydryl reagent 2-sulfonatoethyl-methanethiosulfonate (MTSES), whereas the activity of a mutant AAC2, devoid of cysteines, is unaffected. Wild-type and cysteine-less AAC2 were coexpressed in different molar ratios in yeast mitochondrial membranes. After addition of MTSES the residual transport activity correlated linearly with the fraction of cysteine-less carrier present in the membranes, and so the two versions functioned independently of each other. Also, the cysteine-less and wild-type carriers were purified separately, mixed in defined ratios and reconstituted into liposomes. Again, the residual transport activity in the presence of MTSES depended linearly on the amount of cysteine-less carrier. Thus, the entire transport cycle for ADP/ATP exchange is carried out by the monomer.

Estimation of structural similarity of membrane proteins by hydropathy profile alignment

Many membrane proteins consist of bundles of alpha-helices that are reflected in typical hydropathy profiles of the amino acid sequences. The profiles provide a link between the amino acid sequence of the polypeptide chain and its folding and are much better conserved during evolution than the amino acid sequences from which they are deduced. In this paper, the hydropathy profiles are used to compare structures of membrane proteins or families of membrane proteins. A technique is proposed that computes the optimal alignment of hydropathy profiles without making use of the underlying sequences. The results show that two membrane proteins with only marginal sequence identity or two non-related families of membrane proteins can have very similar hydropathy profiles, indicating similar global structures. Two parameters are defined that measure differences between hydropathy profiles. The Structure Divergence Score (SDS) provides a measure for the divergence in profiles that reflect one and the same global structure. The SDS is derived from the individual hydropathy profiles of the members of a homologous protein family that are believed to share the same structure. The Profile Difference Score (PDS) quantifies the difference between two hydropathy profiles. Comparison of the PDS of the optimal alignment of the hydropathy profiles of two families of membrane proteins with the SDSs of the two families provides a criterion for structural similarity. Using this technique, pairwise alignment of the family profiles of eight families of secondary transporters suggests that the families fall into four structural classes.