Publications

Most proteins start to fold cotranslationally as they come off the ribosome. So far, studies of cotranslational folding have focused mainly on small, single-domain proteins. Here, we have used Force Profile Analysis to study the cotranslational folding of Firefly luciferase, a complex 550-residue protein composed of an N-terminal domain (NTD) encompassing two split Rossmann folds (RF-1, RF-2) and a β-roll, and a flexibly attached C-terminal domain (CTD). The folding process is characterized by a quasi-continuous series of compaction/folding steps that generate intermediate-size pulling forces on the nascent chain, punctuated by a prominent high-force event that represents the folding of the RF-2 domain, and a few low-force instances that likely indicate the formation of distinct folding intermediates. Trigger Factor interacts extensively with the nascent chain when the central part of RF-2 and the early parts of the CTD are synthesized. Our analysis uncovers a cotranslational compaction/folding process that is rich in detail and not just a simple succession of a few distinct, cooperative folding transitions.

We present an approach that allows rapid determination of the topology of Escherichia coli inner-membrane proteins by a combination of topology prediction and limited fusion-protein analysis. We derive new topology models for 12 inner-membrane proteins: MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA. We estimate that our approach should make it possible to arrive at highly reliable topology models for roughly 10% of the ≈800 inner-membrane proteins thought to exist in E. coli .

Recent work has demonstrated that cotranslational folding of proteins or protein domains in, or in the immediate vicinity of, the ribosome exit tunnel generates a pulling force on the nascent polypeptide chain that can be detected using a so-called translational arrest peptide (AP) engineered into the nascent chain as a force sensor. Here, we show that AP-based force measurements combined with systematic Ala and Trp scans of a zinc-finger domain that folds in the exit tunnel can be used to identify the residues that are critical for intraribosomal folding. Our results suggest a general approach to characterize the folded state(s) that may form as a protein domain moves progressively down the ribosome exit tunnel.

The divisome is the macromolecular complex that carries out cell division in Escherichia coli. Every generation it must be assembled, and then disassembled so that the sequestered proteins can be recycled. Whilst the assembly process has been well studied, virtually nothing is known about the disassembly process. In this study, we have used super-resolution SIM imaging to monitor pairs of fluorescently tagged divisome proteins as they depart from the division septum. These simple binary comparisons indicated that disassembly occurs in a coordinated process that consists of at least five steps: [FtsZ, ZapA] ⇒ [ZipA, FtsA] ⇒ [FtsL, FtsQ] ⇒ [FtsI, FtsN] ⇒ [FtsN]. This sequence of events is remarkably similar to the assembly process, indicating that disassembly follows a first-in, first-out principle. A secondary observation from these binary comparisons was that FtsZ and FtsN formed division rings that were spatially separated throughout the division process. Thus the data indicate that the divisome structure can be visualized as two concentric rings; a proto-ring containing FtsZ and an FtsN-ring.

Human growth hormone (hGH) is a four-helix bundle protein of considerable pharmacological interest. Recombinant hGH is produced in bacteria, yet little is known about its folding during expression in E. coli . We have studied the cotranslational folding of hGH using Force Profile Analysis (FPA), both during in vitro translation in the absence and presence of the chaperone trigger factor (TF), and when expressed in E. coli . We find that the main folding transition starts before hGH is completely released from the ribosome, and that it can interact with TF and possibly other chaperones.

No abstract is provided for this article.

Proteins interacting with membranes via a single hydrophobic segment can be classified as either monotopic or bitopic. Here, we probe the topology of a membrane-attached enzyme, the ∈ isoform of human diacylglycerol kinase (DGK∈), when inserted into rough microsomes and compare it with the monotopic membrane protein mouse caveolin-1. In contrast to previous findings, the N-terminal hydrophobic stretch in DGK∈ attains a bitopic rather than a monotopic topology in our experimental system. In addition, we find that charged flanking residues as well as proline residues embedded in the hydrophobic segment are important determinants of monotopic versus bitopic topology.

α-Helix formation in globular proteins has been studied both theoretically and experimentally for decades, while a lack of both high-resolution structures and suitable experimental techniques has hampered the study of helices in membrane proteins. We have developed a new experimental approach, glycosylation mapping, where the active site of the lumenally exposed endoplasmic reticulum enzyme oligosaccharyl transferase is used as a point of reference against which the position of a transmembrane segment in the membrane can be measured. Here, we report an initial analysis of the helix-breaking properties of proline residues inserted in a transmembrane helix. We find that proline residues can break a transmembrane helix, but only when inserted near the end, and only when the helix is sufficiently long. The glycosylation mapping technique may be generally useful for determining the position of transmembrane helices in the membrane.

A priori, single residue insertions into transmembrane helices are expected to be highly disruptive to protein structure and function. We have carried out a systematic analysis of the phenotypes associated with Ala insertions into transmembrane helices in lactose permease, a multispanningEscherichia coli inner membrane protein. Insertion of alanine into the center of 7 transmembrane helices was found to abolish stable integration of lactose permease into the membrane or uphill lactose transport. A more detailed Ala insertion scan was made of transmembrane helix III. The results pin-point a central region of ∼2 helical turns that is crucial for lactose permease stability and/or activity. A Trp scan in this region identified 2 residues essential for lactose permease stability. From these results, it appears that transmembrane helices have differential sensitivities to single residue insertions and that such mutations may be useful for identifying structurally and/or functionally important helix segments.