Publications

Sec dependent and sec independent assembly of E. coli inner membrane proteins: the topological rules depend on chain length.

No abstract is provided for this article.

Membrane Protein Structure: Prediction versus Reality

Since high-resolution structural data are still scarce, different kinds of theoretical structure prediction algorithms are of major importance in membrane protein biochemistry. But how well do the current prediction methods perform? Which structural features can be predicted and which cannot? And what can we expect in the next few years?

Insertion of short transmembrane helices by the Sec61 translocon

The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.

Cotranslational folding of alkaline phosphatase in the periplasm of <scp><i>Escherichia coli</i></scp>

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.

Prediction of the human membrane proteome

Membrane proteins are key molecules in the cell, and are important targets for pharmaceutical drugs. Few three‐dimensional structures of membrane proteins have been obtained, which makes computational prediction of membrane proteins crucial for studies of these key molecules. Here, seven membrane protein topology prediction methods based on different underlying algorithms, such as hidden Markov models, neural networks and support vector machines, have been used for analysis of the protein sequences from the 21 416 annotated genes in the human genome. The number of genes coding for a protein with predicted α‐helical transmembrane region(s) ranged from 5508 to 7651, depending on the method used. Based on a majority decision method, we estimate 5539 human genes to code for membrane proteins, corresponding to approximately 26% of the human protein‐coding genes. The largest fraction of these proteins has only one predicted transmembrane region, but there are also many proteins with seven predicted transmembrane regions, including the G‐protein coupled receptors. A visualization tool displaying the topologies suggested by the eight prediction methods, for all predicted membrane proteins, is available on the public Human Protein Atlas portal ( www.proteinatlas.org ).

Calmodulin assists during co‐translational folding of the K V 7 .2 channel calcium responsive domain

In vivo, the majority of nascent protein chains must begin folding during translation in order to obtain their native structure. While the importance of co-translational folding has become increasingly clear, the specific mechanisms underlying the coordination between the ribosome, nascent chain and molecular chaperones are still uncertain. Here, we have constructed a model of the co-translational folding pathway of the calcium responsive domain (CRD) of the human neuronal KV7.2 human neuronal ion channel, showing that calmodulin (CaM) is crucial. By combining Force Profile Analysis and single-molecule force spectroscopy techniques, we found that CaM, in a calcium-dependent manner, affects early folding events involving three key α-helices in the CRD. In addition, this study suggests that CaM at early stages induces the formation of metastable hairpins, as a part of the co-translational folding pathway. These findings expand on the role of CaM as a key regulator of folding in eukaryotes: not only as an essential cellular signaling protein, but also as a bona fide co-translational molecular chaperone.

Estimating Z‐ring radius and contraction in dividing <i>Escherichia coli</i>

We present a fluorescence recovery after photobleaching-based method for monitoring the progression of septal Z-ring contraction in dividing Escherichia coli cells. In a large number of cells undergoing division, we irreversibly bleached cytosolically expressed Enhanced Green Fluorescent Protein on one side of the septal invagination and followed the fluorescence relaxation on both sides of the septum. Since the relaxation time depends on the cross-sectional area of the septum, it can be used to determine the septal radius r. Assuming that the fraction of the observed cells with r-values in a given interval reflects the duration of that interval in the division process we could derive an approximate time-course for the contraction event, as a population average. By applying the method repeatedly on individual cells, the contraction process was also followed in real time. On a population average level, our data are best described by a linear contraction process in time. However, on the single cell level the contraction processes display a complex behaviour, with varying levels of activity. The proposed approach provides a simple yet versatile method for studying Z-ring contraction in vivo, and will help to elucidate its underlying mechanisms.

Interactions of Peptides with Lipid Bilayers

No abstract is provided for this article.

Preface

Experimentally based topology models for <i>E. coli</i> inner membrane proteins

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.

Bioinformatics of Membrane Proteins

No abstract is provided for this article.

Positional editing of transmembrane domains during ion channel assembly

Summary The integration of transmembrane (TM)-spanning regions of many channels and ion transporters is potentially compromised by the presence of polar and charged residues required for biological function. Although the two TMs of the ATP-gated ion channel subunit P2X2 each contain charged/polar amino acids, we found that each TM is efficiently membrane inserted when it is analysed in isolation, and uncovered no evidence for cooperativity between these two TMs during P2X2 integration. However, using minimal N-glycosylation distance mapping, we find that the positioning of TM2 in newly synthesized P2X2 monomers is distinct from that seen in subunits of the high-resolution structures of assembled homologous trimers. We conclude that P2X2 monomers are initially synthesised at the endoplasmic reticulum in a distinct conformation, where the extent of the TM-spanning regions is primarily defined by the thermodynamic cost of their membrane integration at the Sec61 translocon. In this model, TM2 of P2X2 subsequently undergoes a process of positional editing within the membrane that correlates with trimerisation of the monomer, a process requiring specific polar/charged residues in both TM1 and TM2. We postulate that the assembly process offsets any energetic cost of relocating TM2, and find evidence that positional editing of TM2 in the acid-sensing ion channel (ASIC1a) is even more pronounced than that observed for P2X2. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains. We propose that the orchestrated repositioning of TM segments during subunit oligomerisation plays an important role in generating the functional architecture of active ion channels, and suggest that the regulation of this underappreciated biosynthetic step may provide an elegant mechanism for maintaining ER homeostasis.

Molten globules lure transmembrane helices away from the membrane

Thanks to the rapid progress in membrane protein crystallography and cryogenic electron microscopy in recent years, we have become increasingly accustomed to seeing protein structures characterized by long transmembrane α-helices (TMHs) crisscrossing a lipid bilayer (1). But how is it decided which segments of the polypeptide form the TMHs, and what are the precise sequence requirements for their membrane insertion? The great majority of integral membrane proteins require one or another type of translocon to catalyze insertion into the target membrane. The most commonly used and best understood of these are the universally conserved Sec-type translocons found in the cytoplasmic membrane of bacteria (the SecYEG complex) and the endoplasmic reticulum (ER) membrane in eukaryotes (the Sec61 complex) (2). They mediate cotranslational membrane insertion of the growing polypeptide as it emerges from the exit tunnel of translocon-bound ribosomes (3), and have an evolutionary conserved structure with a central channel through which hydrophilic polypeptide segments can be translocated across the membrane and a dynamic “lateral gate” through which hydrophobic segments in the nascent polypeptide chain can partition into the surrounding lipid bilayer to form TMHs (4, 5). Over the past two decades, statistical and experimental studies have progressively uncovered the sequence characteristics that define the difference between polypeptide segments that can form TMHs and those that cannot. To a first approximation, the efficiency of membrane insertion of a TMH is dictated by its hydrophobicity, although interactions with neighboring TMHs, or sequence variations in segments immediately flanking the TMH, can shift the hydrophobicity threshold (HT) required for … [↵][1]1Email: gunnar{at}dbb.su.se. [1]: #xref-corresp-1-1

Membrane proteins: from bench to bits

Membrane proteins currently receive a lot of attention, in large part thanks to a steady stream of high-resolution X-ray structures. Although the first few structures showed proteins composed of tightly packed bundles of very hydrophobic more or less straight transmembrane α-helices, we now know that helix-bundle membrane proteins can be both highly flexible and contain transmembrane segments that are neither very hydrophobic nor necessarily helical throughout their lengths. This raises questions regarding how membrane proteins are inserted into the membrane and fold in vivo, and also complicates life for bioinformaticians trying to predict membrane protein topology and structure.

How proteins adapt to a membrane–water interface

Membrane proteins present a hydrophobic surface to the surrounding lipid, whereas portions protruding into the aqueous milieu expose a polar surface. But how have proteins evolved to deal with the complex environment at the membrane–water interface? Some insights have been provided by high-resolution structures of membrane proteins, and recent studies of the role of individual amino acids in mediating protein–lipid contacts have shed further light on this issue. It now appears clear that the polar-aromatic residues Trp and Tyr have a specific affinity for a region near the lipid carbonyls, whereas positively charged residues extend into the lipid phosphate region.

<i>Saccharomyces cerevisiae</i> mitochondria lack a bacterial‐type Sec machinery

The bacterial Sec genes encode a generalized protein export machinery. Although the mitochondria present in eukaryotic cells are derived from bacterial ancestors, a comprehensive search of the complete genomic sequence for the eukaryotic yeast Saccharomyces cerevisiae did not reveal any close homologs of the bacterial Sec genes, strongly suggesting that yeast mitochondria lack a generalized bacterial‐type export system. This finding has implications for the sorting of imported mitochondrial proteins to the intermembrane space compartment, and also for the insertion of mitochondrially encoded proteins into the inner membrane.

The Collector's Dream: From Dayhoff to Data Banks

No abstract is provided for this article.

Different positively charged amino acids have similar effects on the topology of a polytopic transmembrane protein in Escherichia coli.

Integral membrane proteins from a wide variety of sources conform to a positive-inside rule, with many more positively charged amino acids in their cytoplasmic as compared to extracytoplasmic domains. A growing body of experimental work also points to positively charged residues in regions flanking the apolar transmembrane segments as being the main topological determinants. In this paper, we report a systematic comparison of the effects of positively (Arg, Lys, His) as well as negatively (Asp, Glu) charged residues on the membrane topology of a model Escherichia coli inner membrane protein. Our results show that positive charge is indeed the major factor determining the transmembrane topology, with Arg and Lys being of nearly equal efficiency. His, although normally a very weak topological determinant, can be potentiated by a lowering of the cytoplasmic pH. Asp and Glu affect the topology to similar extents and only when present in very high numbers.

Nobel Committee responds to criticism

No abstract is provided for this article.