Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Site-specific collapse dynamics guide the formation of the cytochrome c' four-helix bundle.

ABSTRACT: The evolution of tryptophan-to-heme (W/heme) distance distributions extracted from analysis of fluorescence energy transfer kinetics during the refolding of Rhodopseudomonas palustris cytochrome c' reveals dramatic differences between two variants [W32 (Q1A/F32W/W72F) and W72 (Q1A)]. Both W32/heme and W72/heme distance distributions measured at the earliest time point attainable with a continuous-flow mixer (150 mus) confirm that the polypeptide ensemble is not uniformly collapsed and that native structure is not formed. Time-resolved fluorescence spectra indicate that W32 is sequestered from the aqueous solution during the first 700 mus of folding, whereas W72 remains exposed to solvent. The first moment of the W32/heme distance distribution evolves to its native value faster than that of W72, suggesting that the approach of W32 to the heme precedes that of W72.

SUBMITTER: Kimura T

PROVIDER: S-EPMC1765420 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Site-specific collapse dynamics guide the formation of the cytochrome c' four-helix bundle.

Kimura Tetsunari T Lee Jennifer C JC Gray Harry B HB Winkler Jay R JR

Proceedings of the National Academy of Sciences of the United States of America 20061219 1

The evolution of tryptophan-to-heme (W/heme) distance distributions extracted from analysis of fluorescence energy transfer kinetics during the refolding of Rhodopseudomonas palustris cytochrome c' reveals dramatic differences between two variants [W32 (Q1A/F32W/W72F) and W72 (Q1A)]. Both W32/heme and W72/heme distance distributions measured at the earliest time point attainable with a continuous-flow mixer (150 mus) confirm that the polypeptide ensemble is not uniformly collapsed and that nativ ...[more]

PMID: 17179212

Similar Datasets

Cytochrome b562 folding triggered by electron transfer: approaching the speed limit for formation of a four-helix-bundle protein.

Project description:Ferrocytochrome b562 [Fe(II)cyt b562] folding can be triggered by photoinduced electron transfer to unfolded Fe(III)cyt b562 in 2-3 M guanidine hydrochloride solutions. The folding rates increase with decreasing guanidine hydrochloride; the extrapolated time constant for this folding process in the absence of denaturant (5 micros) is near the predicted value for intrachain diffusion. The relatively smooth energy landscape indicated for Fe(II)cyt b562 folding accords with the helical, highly symmetrical structure of the protein.

| S-EPMC21958 | biostudies-literature

A four-helix bundle stores copper for methane oxidation.

Project description:Methane-oxidizing bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase. Certain methanotrophs are also able to switch to using the iron-containing soluble methane monooxygenase to catalyse methane oxidation, with this switchover regulated by copper. Methane monooxygenases are nature's primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and methane monooxygenases have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. Here we discover and characterize a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for particulate methane monooxygenase. Csp1 is a tetramer of four-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realized. Cytosolic homologues of Csp1 are present in diverse bacteria, thus challenging the dogma that such organisms do not use copper in this location.

| S-EPMC4561512 | biostudies-literature

Cytochrome c' folding triggered by electron transfer: fast and slow formation of four-helix bundles.

Project description:Reduced (Fe(II)) Rhodopseudomonas palustris cytochrome c' (Cyt c') is more stable toward unfolding ([GuHCl](1/2) = 2.9(1) M) than the oxidized (Fe(III)) protein ([GuHCl](1/2) = 1.9(1) M). The difference in folding free energies (Delta Delta G(f) degrees = 70 meV) is less than half of the difference in reduction potentials of the folded protein (100 mV vs. NHE) and a free heme in aqueous solution ( approximately -150 mV). The spectroscopic features of unfolded Fe(II)-Cyt c' indicate a low-spin heme that is axially coordinated to methionine sulfur (Met-15 or Met-25). Time-resolved absorption measurements after CO photodissociation from unfolded Fe(II)(CO)-Cyt c' confirm that methionine can bind to the ferroheme on the microsecond time scale [k(obs) = 5(2) x 10(4) s(-1)]. Protein folding was initiated by photoreduction (two-photon laser excitation of NADH) of unfolded Fe(III)-Cyt c' ([GuHCl] = 2.02--2.54 M). Folding kinetics monitored by heme absorption span a wide time range and are highly heterogeneous; there are fast-folding ( approximately 10(3) s(-1)), intermediate-folding (10(2)-10(1) s(-1)), and slow-folding (10(-1) s(-1)) populations, with the last two likely containing methionine-ligated (Met-15 or Met-25) ferrohemes. Kinetics after photoreduction of unfolded Fe(III)-Cyt c' in the presence of CO are attributable to CO binding [1.4(6) x 10(3) s(-1)] and Fe(II)(CO)-Cyt c' folding [2.8(9) s(-1)] processes; stopped-flow triggered folding of Fe(III)-Cyt c' (which does not contain a protein-derived sixth ligand) is adequately described by a single kinetics phase with an estimated folding time constant of approximately 4 ms [Delta G(f) degrees = -33(3) kJ mol(-1)] at zero denaturant.

| S-EPMC35415 | biostudies-literature

Four-alpha-helix bundle with designed anesthetic binding pockets. Part II: halothane effects on structure and dynamics.

Project description:As a model of the protein targets for volatile anesthetics, the dimeric four-alpha-helix bundle, (Aalpha(2)-L1M/L38M)(2), was designed to contain a long hydrophobic core, enclosed by four amphipathic alpha-helices, for specific anesthetic binding. The structural and dynamical analyses of (Aalpha(2)-L1M/L38M)(2) in the absence of anesthetics (another study) showed a highly dynamic antiparallel dimer with an asymmetric arrangement of the four helices and a lateral accessing pathway from the aqueous phase to the hydrophobic core. In this study, we determined the high-resolution NMR structure of (Aalpha(2)-L1M/L38M)(2) in the presence of halothane, a clinically used volatile anesthetic. The high-solution NMR structure, with a backbone root mean-square deviation of 1.72 A (2JST), and the NMR binding measurements revealed that the primary halothane binding site is located between two side-chains of W15 from each monomer, different from the initially designed anesthetic binding sites. Hydrophobic interactions with residues A44 and L18 also contribute to stabilizing the bound halothane. Whereas halothane produces minor changes in the monomer structure, the quaternary arrangement of the dimer is shifted by about half a helical turn and twists relative to each other, which leads to the closure of the lateral access pathway to the hydrophobic core. Quantitative dynamics analyses, including Modelfree analysis of the relaxation data and the Carr-Purcell-Meiboom-Gill transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange both near and remote from the binding site. Our results revealed a novel mechanism of an induced fit between anesthetic molecule and its protein target, with the direct consequence of protein dynamics changing on a global rather than a local scale. This mechanism may be universal to anesthetic action on neuronal proteins.

| S-EPMC2480694 | biostudies-literature

Rational thermostabilisation of four-helix bundle dimeric de novo proteins.

Project description:The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

| S-EPMC8024369 | biostudies-literature

?-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle.

Project description:Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). ?-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that ?-SNAP also dramatically enhances SNARE assembly and membrane fusion. How ?-SNAP is involved in these opposing activities is not known. Here, we examine the effect of ?-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that ?-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, ?-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that ?-SNAP barely bound the partially assembled trans-SNARE complex. Thus, ?-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD.

| S-EPMC4838522 | biostudies-literature

Computational de novo design of a four-helix bundle protein--DND_4HB.

Project description:The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple ?-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD?=?1.11 Å) and >90% of the core side chain ?1 and ?2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices.

| S-EPMC4380976 | biostudies-literature

Four-helix bundle: a ubiquitous sensory module in prokaryotic signal transduction.

Project description:MotivationTransmembrane chemoreceptors in Escherichia coli utilize ligand-binding domains for detecting various external signals. The structure of this domain in the E.coli aspartate receptor, Tar, is known and its signal transduction mechanism is under investigation. Current domain models for this important sensory module are inaccurate and, therefore, cannot reveal the distribution of this domain within the current genomic landscape.ResultsWe carried out sensitive and exhaustive PSI-BLAST searches initiated with the sequence corresponding to a known structure of the four-helix, ligand-binding domain of the aspartate chemoreceptor. From the resulting sequences, we built a multiple sequence alignment for this domain family, which confirmed that the current TarH model is erroneous and fails to detect most of the domain homologs. In the process, we developed a technique that visualizes the secondary structure prediction of each protein sequence in order to improve the multiple sequence alignment. We found that the four-helix up-and-down bundle represents a large domain family and includes representatives of all major classes of prokaryotic signal transduction, namely histidine kinases, di-guanylate cyclases and chemotaxis receptors.

| S-EPMC5073874 | biostudies-literature

De novo design of a transmembrane Zn²⁺-transporting four-helix bundle.

Project description:The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn(2+) and Co(2+), but not Ca(2+), across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn(2+) ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties.

| S-EPMC4400864 | biostudies-literature

Artificial Diiron Enzymes with a De Novo Designed Four-Helix Bundle Structure.

Project description:A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal-binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four-helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron-oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

| S-EPMC5019575 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data