Secondary literature sources for VHP
The following references were automatically generated.
- Choe SE, Li L, Matsudaira PT, Wagner G, Shakhnovich EI
- Differential stabilization of two hydrophobic cores in the transition state of the villin 14T folding reaction.
- J Mol Biol. 2000; 304: 99-115
- Display abstract
We report the distribution of hydrophobic core contacts during the folding reaction transition state for villin 14T, a small 126-residue protein domain. The solution structure of villin 14T contains a central beta-sheet with two flanking hydrophobic cores; transition states for this protein topology have not been previously studied. Villin 14T has no disulfide bonds or cis-proline residues in its native state; it folds reversibly, and in an apparently two-state manner under some conditions. To map the hydrophobic core contacts in the transition state, 27 point mutations were generated at positions spread throughout the two hydrophobic cores. After each point mutation, comparison of the change in folding kinetics with the equilibrium destabilization indicates whether the site of mutation is stabilized in the transition state. The results show that the folding nucleus, or the sub-region with the strongest transition state contacts, is located in one of the two hydrophobic cores (the predominantly aliphatic core). The other hydrophobic core, which is mostly aromatic, makes much weaker contacts in the transition state. This work is the first transition state mapping for a protein with multiple major hydrophobic cores in a single folding unit; the hydrophobic cores cannot be separated into individual folding subdomains. The stabilization of only one hydrophobic core in the transition state illustrates that hydrophobic core formation is not intrinsically capable of nucleating folding, but must also involve the right specific interactions or topological factors in order to be kinetically important.
- Klahre U, Friederich E, Kost B, Louvard D, Chua NH
- Villin-like actin-binding proteins are expressed ubiquitously in Arabidopsis.
- Plant Physiol. 2000; 122: 35-48
- Display abstract
In an attempt to elucidate the biological function of villin-like actin-binding proteins in plants we have cloned several genes encoding Arabidopsis proteins with high homology to animal villin. We found that Arabidopsis contains at least four villin-like genes (AtVLNs) encoding four different VLN isoforms. Two AtVLN isoforms are more closely related to mammalian villin in their primary structure and are also antigenically related, whereas the other two contain significant changes in the C-terminal headpiece domain. RNA and promoter/beta-glucuronidase expression studies demonstrated that AtVLN genes are expressed in all organs, with elevated expression levels in certain types of cells. These results suggest that AtVLNs have less-specialized functions than mammalian villin, which is found only in the microvilli of brush border cells. Immunoblot experiments using a monoclonal antibody against pig villin showed that AtVLNs are widely distributed in a variety of plant tissues. Green fluorescent protein fused to full-length AtVLN and individual AtVLN headpiece domains can bind to both animal and plant actin filaments in vivo.
- Gonzalez-Barroso MM et al.
- Structural and functional study of a conserved region in the uncoupling protein UCP1: the three matrix loops are involved in the control of transport.
- J Mol Biol. 1999; 292: 137-49
- Display abstract
It has been reported that the region 261-269 of the uncoupling protein from brown adipose tissue mitochondria, UCP1, has an important role in the control of its proton translocating activity. Thus the deletion of residues Phe267-Lys268-Gly269 leads to the loss of the nucleotide regulation of the protein, while the complete deletion of the segment leads to the formation of a pore. The region displays sequence homology with the DNA-binding domain of the estrogen receptor. The present report analyzes the structure, by NMR and circular dichroism, of a 20 amino acid residue peptide containing the residues of interest. We demonstrate that residues 263-268 adopt an alpha-helical structure. The helix is at the N-terminal end of the sixth transmembrane domain. The functional significance of this helix has been examined by site-directed mutagenesis of the protein expressed recombinantly in yeasts. Alterations in the structure or orientation of the region leads to an impairment of the regulation, by nucleotides and fatty acids, of the transport activity. UCP1 is one member of the family formed by the carriers of the mitochondrial inner membrane. The family is characterized by a tripartite structure with three repeated segments of about 100 amino acid residues. Two of the mutations have also been performed in the first and second matrix loops and the effect on UCP1 function is very similar. We conclude that the three matrix loops contribute to the formation of the gating domain in UCP1 and propose that they form a hydrophobic pocket that accommodates the purine moiety of the bound nucleotide.
- Matova N, Mahajan-Miklos S, Mooseker MS, Cooley L
- Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells.
- Development. 1999; 126: 5645-57
- Display abstract
Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.
- Roof DJ, Hayes A, Adamian M, Chishti AH, Li T
- Molecular characterization of abLIM, a novel actin-binding and double zinc finger protein.
- J Cell Biol. 1997; 138: 575-88
- Display abstract
Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is approximately 50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments.
- Markus MA, Matsudaira P, Wagner G
- Refined structure of villin 14T and a detailed comparison with other actin-severing domains.
- Protein Sci. 1997; 6: 1197-209
- Display abstract
Villin 14T is the amino terminal actin monomer binding domain from the actin-severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin-severing proteins. Protein Science 3:70-81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin-severing domain, severin domain 2.
- Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ
- Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.
- J Cell Biol. 1997; 139: 1255-69
- Display abstract
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.
- McKnight CJ, Doering DS, Matsudaira PT, Kim PS
- A thermostable 35-residue subdomain within villin headpiece.
- J Mol Biol. 1996; 260: 126-34
- Display abstract
The actin-bundling protein villin contains, at its extreme C terminus, a compact f-actin binding domain called "headpiece". This 76-amino acid domain from chicken is highly thermostable. Here, we show that the stable folded structure in headpiece is localized to a subdomain formed by the C-terminal 35 residues. The subdomain, denoted HP-35, is monomeric and retains high thermostability, with a Tm of 70( +/- 1) degree C at PH 7.0. There are no cysteine residues in HP-35 and its folding is not dependent on the binding of metals or other ligands. HP-35 is not a molten globule, but instead, has properties expected for a fully folded protein with a unique structure. In particular, the slowly exchanging amide protons in HP-35 have protection factors that are slightly larger than those predicted if exchange occurred only from globally unfolded molecules. NMR studies indicate that the headpiece subdomain contains three short alpha-helices, and that these same helices are present in the corresponding regions of intact headpiece. HP-35 is the smallest monomeric polypeptide characterized consisting of only naturally occurring amino acids that autonomously folds into a unique and thermostable structure without disulfide bonds or ligand binding.
- Simenel C et al.
- Conformational behaviour of a synthetic peptide of the C-terminus of villin that interacts with actin: an NMR, CD and stimulated annealing study.
- Int J Pept Protein Res. 1995; 45: 574-86
- Display abstract
The solution structure of a synthetic 22-amino acid peptide (P1) corresponding to the extreme C-terminal end and one of the F-actin binding sites of villin has been determined by 1H NMR and CD spectroscopy. The structure of this peptide was compared to that of a peptide in which lysine to glutamic acid substitutions were introduced at positions 17 and 19 (P11), abolishing F-actin binding. Both peptides are largely unstructured in aqueous solution. Changes observed in the NMR and CD spectra of both peptides are consistent with alpha-helix formation in trifluoroethanol/water mixtures. A set of 189 interproton distances derived from nuclear Overhauser enhancement (NOE) measurements, 17 phi-angle constraints obtained from 3JNH alpha coupling constants, as well as about 10 N ... O distance restraints deduced from amide proton exchange kinetics with deuterium, were used for the structure determination. The three-dimensional structure of P1 and P11 is characterized by two helical regions, one extending from residues 2 to 5 and a second covering residues 7 to 17. The central fragment, ranging from Leu-7 to Leu-15, is more stable. The C-terminal residues are less structured, particularly within peptide P11. The significance of these structural results is discussed in relation to the biological activity of villin.
- Azim AC, Knoll JH, Beggs AH, Chishti AH
- Isoform cloning, actin binding, and chromosomal localization of human erythroid dematin, a member of the villin superfamily.
- J Biol Chem. 1995; 270: 17407-13
- Display abstract
Dematin is an actin-bundling protein of the erythroid membrane skeleton and is abundantly expressed in human brain, heart, skeletal, muscle, kidney, and lung. The 48-kDa subunit of dematin contains a headpiece domain which was originally identified in villin, and actin-binding protein of the brush-border cytoskeleton. The head-piece domain of villin is essential for its morphogenic function in vivo. Here we report the primary structure of 52-kDa subunit of dematin which differs from the 48-kDa subunit by a 22-amino-acid insertion within its headpiece domain. A unique feature of the insertion sequence of the 52-kDa subunit is its homology to erythrocyte protein 4.2. The insertion sequence also includes a cysteine residue which may explain the formation of sulfhydryl-linked trimers of dematin. Actin binding measurements using recombinant fusion proteins revealed that each monomer of dematin contains two F-actin binding sites: one in the headpiece domain and the other in the undefined N-terminal domain. Although the actin bundling activity of intact dematin was abolished by phosphorylation, no effect of phosphorylation was observed on the actin binding activity of fusion proteins. Using somatic cell hybrid panels and fluorescence in situ hybridization, the dematin gene was localized on the short arm of chromosome 8. The dematin locus, 8p21.1, is distal to the known locus of human erythroid ankyrin (8p11.2) and may contribute to the etiology of hemolytic anemia in a subset of patients with severe hereditary spherocytosis.
- Finidori J, Friederich E, Kwiatkowski DJ, Louvard D
- In vivo analysis of functional domains from villin and gelsolin.
- J Cell Biol. 1992; 116: 1145-55
- Display abstract
Transfected CV1 cells were used to compare the in vivo effects of various domains of villin and gelsolin. These two homologous actin modulating proteins both contain a duplicated severin-like sequence. Villin has in addition a carboxy-terminal domain, the headpiece, which accounts for its bundling activity. The effects of the villin-deleted mutants were compared with those of native villin. Our results show that essential domains of villin required to induce the growth of microvilli and F-actin redistribution are present in the first half of the core and in the headpiece. We also show that the second half of the villin core cannot be exchanged by its homolog in gelsolin. When expressed at high levels of CV1 cells, full length gelsolin completely disrupted stress fibers without change of the cell shape. Addition of the villin headpiece to gelsolin had no effect on the phenotype induced by gelsolin alone. Expression of the first half of gelsolin induced similar modifications as capping proteins and rapid cell mortality; this deleterious effect on the cell structure was also observed when the headpiece was linked to the first half of gelsolin. In cells expressing the second half of gelsolin, a dotted F-actin staining was often seen. Moreover elongated dorsal F-actin structures were observed when the headpiece was linked to the second gelsolin domain. These studies illustrate the patent in vivo severing activity of gelsolin as well as the distinct functional properties of villin core in contrast to gelsolin.
- Matsudaira P, Jakes R, Cameron L, Atherton E
- Mapping the cysteine residues and actin-binding regions of villin by using antisera to the amino and carboxyl termini of the molecule.
- Proc Natl Acad Sci U S A. 1985; 82: 6788-92
- Display abstract
Peptide antisera specific for either the amino- or carboxyl-terminal regions of villin were used to locate the position of cysteine residues in immunoblots of villin cleaved with 2-nitro-5-thiocyanobenzoic acid. Maps constructed from the cleavage pattern suggest that villin contains six cysteine residues, two located in its amino-terminal peptide of Mr 44,000, and four located in the carboxyl-terminal peptide of Mr 51,000. Gel overlays of the partial cleavage fragments with 125I-labeled actin identified a calcium-dependent actin-binding region located within the amino-terminal peptide of Mr 32,000 of villin. The peptide antibody method used, called cleavage mapping, should be a convenient technique for mapping residues and ligand binding sites in proteins.
- Hesterberg LK, Weber K
- Ligand-induced conformational changes in villin, a calcium-controlled actin-modulating protein.
- J Biol Chem. 1983; 258: 359-64
- Display abstract
The physical structure of villin, a Ca2+-modulated regulator protein of actin filament organization, has been studied in the absence and presence of Ca2+ using analytical ultracentrifugation, gel chromatography, ultraviolet difference spectroscopy, and circular dichroism. Villin exhibits an intrinsic sedimentation coefficient, S020, w, of 5.0 s and an apparent Stokes radius, Rs, of 44 A in EGTA-containing buffer. In the presence of greater than 20 microM Ca2+, villin shows a S020, w of 4.1 s and Rs of 49 A, indicative of a conformational change in the protein. No significant changes in the partial specific volume (0.73) of villin are observed in the presence of Ca2+, and sedimentation equilibrium studies demonstrates that the effects of Ca2+ are not due to a change in the molecular mass (95,000 daltons). The combined hydrodynamic data suggest that villin is an asymmetric molecule with an axial ratio of 4.5:1, based on a prolate ellipsoid model at 0.5 g/g of hydration, corresponding to a maximum length of 84 A. The presence of Ca2+ changes the shape to a more asymmetric molecule with an axial ratio of 8:1 and a maximum length of 123 A. Since the large proteolytic fragment, villin core, does not exhibit the strong structural change of villin, an important function of the small villin headpiece domain in the observed conformational change of intact villin is suggested. The results indicate new aspects of the function of villin as cross-linker in microvillus core filament bundles and the disintegration of these structures in the presence of calcium.