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Jones, Jonathan A.; Wilkins, Deborah K.; Smith, Lorna J.; Dobson, Christopher M.

doi:10.1023/a:1018304117895

Cited by 213 publications

(135 citation statements)

References 13 publications

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“…These values may correspond to micelle-associated ForC GBD. It should be noted here that the D values for the fully unfolded protein may be in the same range (44). However, based on the CD measurements, the DPC micelle-immersed form is not unfolded but has a high content of secondary structure.…”

Section: Resultsmentioning

confidence: 84%

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Dames

Junemann

Sass

et al. 2011

Journal of Biological Chemistry

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Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitinlike ␣/␤-roll fold with an unusually long loop between ␤-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of ␣-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the ␤1-␤2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-L-serine) and 1,2-dihexanoyl-snglycero-3-phospho-(1 -rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes.Formins are widely expressed multidomain proteins that regulate the spatial and temporal organization of the actin and microtubule cytoskeleton, thereby affecting important cellular functions, such as cytokinesis and cell-cell adhesion (1-3). The defining element is the ϳ400-amino acid-encompassing "formin homology 2" (FH2) 2 domain that can tightly associate with the barbed end of elongating actin filaments (4). In nearly all formins, the FH2 domain is preceded by a prolinerich "formin homology 1" (FH1) region that varies in length and the number of binding sites for profilin and that is used to recruit profilin-actin complexes for filament elongation. The influence of the FH2 domain on the rate of filament elongation depends on the length and composition of the FH1 region and the connecting linker (2, 4, 5). Most formins contain additional regulatory domains. The N-terminal "formin homology 3" (FH3) domain mediates interactions with autoinhibitory regulatory elements in the C terminus as well as with other formin regulators. The FH3 domain can be further divided into functional subdomains, such as an armadill...

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Section: Resultsmentioning

confidence: 84%

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Dames

Junemann

Sass

et al. 2011

Journal of Biological Chemistry

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“…The molecular dimensions of N and MFI species were compared by performing NMR pulsed-field gradient diffusion measurements (12,13,18,19). The approach focused on the estimation of the effective hydrodynamic radii of both species with a pulsed-gradient-stimulated echo longitudinal encode-decode pulse sequence (12,18) in which the protein NMR signal decay is measured as a function of gradient field strength. The signal decay rate is proportional to the diffusion coefficient D, which is itself inversely proportional to the effective hydrodynamic radius, R H .…”

Section: Resultsmentioning

confidence: 99%

“…All NMR data were processed by using FELIX 2.3 software from Biosym Technologies (San Diego). Pulsed-field gradient diffusion measurements were performed with a pulsed gradient stimulated echo longitudinal encode-decode pulse sequence (12,13). The length of pulses and delays was held constant, and 20 spectra were acquired with the strength of the diffusion gradient varying between 5% and 100% of its maximum value; each measurement was repeated six times to improve the signal-to-noise ratio and to allow the measurement error to be estimated.…”

Section: Rp-hplc and Electrospray Ionization-ms (Esi-ms)mentioning

confidence: 99%

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Cemazar

Zahariev

Lopez

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The oxidative folding of the Amaranthus ␣-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by 1 H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.T he cystine knot, or the knottin fold, is a compact structure consisting of a short triple-stranded antiparallel ␤-sheet reinforced by three disulfide bridges that form a topological knot [see Amaranthus ␣-amylase inhibitor (AAI) structure, Fig. 1A Inset]. It is found both in small peptides and as a domain of larger proteins (1) and in addition, it is a recurrent substructure of larger cysteine-rich motifs such as the well known conotoxin fold (2). Despite their small size, cystine knots can fulfill a large variety of functions ranging from ion-channel blocking to enzyme inhibition, which makes them ideal protein engineering scaffolds for industrial applications (3, 4). Because cystine knots occur in many unrelated species (fungi, plants, snails, and spiders, etc.), it is presumed that this fold has emerged via convergent evolution (2). The compactness and versatility of this fold are best illustrated by the fact that both the shortest peptide inhibitor of ␣-amylases, AAI [32 amino acids (Fig. 1 A)] (5-8) and the shortest lectin molecule known so far (9), are members of this family.Cystine knot peptides are known to form readily in vitro under the conditions of oxidative folding (4). In this work, we describe an analysis of the oxidative folding intermediates of AAI, which shows that the native protein (N) forms via several fully oxidized intermediates with nonnative disulfide bonds, including the vicinal disulfide 17-18. NMR spectroscopy revealed that the main folding intermediate has a structure as compact as the completely folded peptide, comprising a number of slowly interconverting backbone conformations. On the basis of the experimental data, we suggest that the compactness, a prerequisi...

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“…These experiments were also recorded with the reconstituted Ig2/70S sample for comparison. 1 H PFG-LED diffusion measurements (34) were also recorded for RNC samples, to monitor the translational diffusion of the 70S ribosome by using the observable proton resonances of the stalk L7/L12 proteins (20). HNCO experiments ( 1 H-15 N or 1 H-13 C correlation projections) were recorded and revealed that the observed 1 H- 15 N correlations in the SOFAST-HMQC spectra could not arise from free amino acids because the presence of a peptide bond is the prerequisite for detection of H i N i CO iϪ1 correlations (35).…”

Section: Methodsmentioning

confidence: 99%

Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

Hsu

Fucini

Cabrita

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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157

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Protein folding in living cells is inherently coupled to protein synthesis and chain elongation. There is considerable evidence that some nascent chains fold into their native structures in a cotranslational manner before release from the ribosome, but, despite its importance, a detailed description of such a process at the atomic level remains elusive. We show here at a residue-specific level that a nascent protein chain can reach its native tertiary structure on the ribosome. By generating translation-arrested ribosomes in which the newly synthesized polypeptide chain is selectively 13 C/ 15 Nlabeled, we observe, using ultrafast NMR techniques, a large number of resonances of a ribosome-bound nascent chain complex corresponding to a pair of C-terminally truncated immunoglobulin (Ig) domains. Analysis of these spectra reveals that the nascent chain adopts a structure in which a native-like N-terminal Ig domain is tethered to the ribosome by a largely unfolded and highly flexible C-terminal domain. Selective broadening of resonances for a group of residues that are colocalized in the structure demonstrates that there are specific but transient interactions between the ribosome and the N-terminal region of the folded Ig domain. These findings represent a step toward a detailed structural understanding of the cellular processes of cotranslational folding.cotranslational folding

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Cited by 213 publications

References 13 publications

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues

Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

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