Publications

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 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.

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.

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.

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.

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.