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ABSTRACT: The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive post-translational secretion of preproteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, "closed" state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more "open", has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1) and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.
Biochemistry 02/2012; 51(7):1369-79. · 3.42 Impact Factor
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ABSTRACT: The conformational plasticity of serine protease inhibitors (serpins) underlies both their activities as protease inhibitors and their susceptibility to pathogenic misfolding and aggregation. Here, we structurally characterize a sheet-opened state of the serpin α-1 antitrypsin (α₁AT) and show how local unfolding allows functionally essential strand insertion. Mutations in α₁AT that cause polymerization-induced serpinopathies map to the labile region, suggesting that the evolution of serpin function required sampling of high risk conformations on a dynamic energy landscape.
Nature Structural & Molecular Biology 02/2011; 18(2):222-6. · 12.71 Impact Factor
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ABSTRACT: In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)(5) and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively.
Journal of Molecular Biology 12/2008; 385(4):1243-56. · 4.00 Impact Factor
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ABSTRACT: We have designed "split tetra-Cys motifs" that bind the biarsenical fluorescein dye 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) across strands of a model beta-rich protein. Our strategy was to divide the linear FlAsH binding tetra-Cys sequence such that dye could be fully liganded only when the strands were arranged in space correctly by native protein conformational proximities. We introduced pairs of alternating cysteines on adjacent beta strands of cellular retinoic acid binding protein to create FlAsH binding sites in the native structure. Selective labeling occurred both in vitro and in vivo relative to sites with fewer than four Cys or with inappropriate geometry. Interestingly, two of the split tetra-Cys motif-carrying proteins bound FlAsH whether native or urea unfolded, while one was capable of binding FlAsH only when native. This latter design exemplifies the potential of split motifs as structure sensors.
Chemistry & Biology 11/2008; 15(10):1104-15. · 5.83 Impact Factor
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ABSTRACT: A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.
Biopolymers 02/2007; 88(2):157-63. · 2.87 Impact Factor
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ABSTRACT: Coupling genetically encoded target sequences with specific and selective labeling strategies has made it possible to utilize fluorescence spectroscopy in complex mixtures to investigate the structure, function, and dynamics of proteins. Thus, there is a growing need for a repertoire of such labeling approaches to deploy based on a given application and to utilize in combination with one another by orthogonal reactivity. We have developed a simple approach to synthesize a fluorescent probe that binds to a poly-histidine sequence. The amino group of cysteine was converted into nitrilotriacetate to create a metal-chelating cysteine molecule, Cys-nitrilotriacetate. Two Cys-nitrilotriacetate molecules were then cross-linked using dibromobimane to generate a fluorophore capable of binding a His-tag on a protein, NTA(2)-BM. NTA(2)-BM is a potential fluorophore for selective tagging of proteins in vivo.
Chemical Biology & Drug Design 02/2007; 69(1):31-40. · 2.28 Impact Factor
