Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle α-tropomyosin in an engineered chimeric protein 1 1Edited by P. E. Wright

Greenfield, Norma J.; Huang, Yuanpeng Janet; Palm, Thomas; Swapna, G.V.T.; Monleón, Daniel; Montelione, Gaetano T.; Hitchcock‐DeGregori, Sarah E.

doi:10.1006/jmbi.2001.4982

Cited by 64 publications

(117 citation statements)

References 75 publications

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“…TM peptides, AcTM1aZip and AcTM1bZip, are designed chimeric proteins that contain 14 residues of long rat α-tropomyosin encoded by exon 1a or 19 residues of short rat α-tropomyosin encoded by exon 1b correspondingly and the 18 C-terminal residues of the GCN4 leucine zipper domain (37); their structures were solved using NMR (38). These peptides and N-acetylated striated muscle α-tropomyosin, stTM, were a gift from Dr. Sarah Hitchcock-DeGregori (RWJMS, Piscataway, NJ).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

2006

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Tropomodulin, a tropomyosin-binding protein, caps the slow-growing (pointed) end of the actin filament regulating its dynamics. Tropomodulin, therefore, is important for determining cell morphology, cell movement, and muscle contraction. For the first time we show that one tropomodulin molecule simultaneously binds two tropomyosin molecules in a cooperative manner. On the basis of the tropomodulin solution structure and predicted secondary structure, we introduced a series of point mutations in regions important for tropomyosin-binding and actin-capping. Capping activity of these mutants was assayed by measuring actin polymerization using pyrene fluorescence. Using direct methods (circular dichroism and native gel-electrophoresis) for detecting tropomodulin/ tropomyosin binding, we localized the second tropomyosin-binding site to residues 109-144. Despite previous reports that the second binding site is for erythrocyte tropomyosin only, we found that both short non-muscle and long muscle α-tropomyosins bind there as well, though with different affinities. We propose a model for actin capping where one tropomodulin molecule can bind to two tropomyosin molecules at the pointed end. Keywordstropomodulin; tropomyosin; actin filament; pointed end Actin is a major component of the cell cytoskeleton and the sarcomeres of striated muscle, and plays a critical role in cell morphology, cell movement, and muscle contraction (1,2). It is known that actin filaments form vastly different structures in different cell types and in different locations within the cell. Regulation of the dynamics at actin's ends is of central importance in the assembly of these various structures. An actin filament has two distinct ends: a fastgrowing barbed end and a slow-growing pointed end (3). The exclusive properties of these ends are exploited by over 160 distinct actin binding proteins, including those that cap, sever, or cross-link filaments (4). Of particular interest is tropomodulin (Tmod), a tropomyosinbinding protein, which regulates actin filament dynamics by capping the pointed end.Tropomyosin (TM) is a two-chained coiled coil that binds head-to-tail (N to C terminus) along the length of both sides of actin filaments, spanning 6-7 actin monomers per TM molecule, depending on the isoform (5). There are a variety of TM isoforms encoded by different genes. The N-terminus of TM, which points towards the pointed end of the actin filament (6), is required for interaction with Tmod (7-9). Best known for its role in muscle contraction (10, 11), TM also regulates the stiffness of actin (12) and influences polymerization at the pointed To whom correspondence should be addressed: Alla S. Kostyukova, end (13,14). Tmod in combination with TM forms a cap at the pointed end, preventing addition or loss of G-actin (15-17).Tropomodulin, originally discovered as a TM-binding protein in erythrocyte membranes (18), is the only known pointed end specific capping protein (15). Presently, there are four known Tmod isoforms (16,19,20). These isofor...

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

2006

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“…43, and rat recombinant ␣-tropomyosins TM2 and TM5a were expressed in E. coli and purified as previously described (37). AcTM1bZip is a designed chimeric protein that contains 19 residues of short rat ␣-tropomyosin encoded by exon 1b and the 18 C-terminal residues of the GCN4 leucine zipper domain (24). It was synthesized by SynPep (Dublin, CA).…”

Section: Constructions Of Expression Vectors Of Tropomodulin N-terminalmentioning

confidence: 99%

“…Tropomyosin isoforms differ in actin affinity and regulatory function and exhibit developmentally regulated and tissue-specific expression patterns (6) as well as different localizations within cells (21). The two major classes of tropomyosin, short (ϳ247 amino acids) and long (ϳ284 amino acids), differ in sequence and structure at the N terminus (22)(23)(24).…”

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confidence: 99%

Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

Kostyukova

Hitchcock‐DeGregori

2004

Journal of Biological Chemistry

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131

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Tropomodulins (Tmod) bind to the N terminus of tropomyosin and cap the pointed end of actin filaments. Tropomyosin alone also inhibits the rate of actin depolymerization at the pointed end of filaments. Here we have defined 1) the structural requirements of the N terminus of tropomyosin important for regulating the pointed end alone and with erythrocyte Tmod (Tmod1), and 2) the Tmod1 subdomains required for binding to tropomyosin and for regulating the pointed end. Changes in pyrene-actin fluorescence during polymerization and depolymerization were measured with actin filaments blocked at the barbed end with gelsolin. Three tropomyosin isoforms differently influence pointed end dynamics. Recombinant TM5a, a short non-muscle ␣-tropomyosin, inhibited depolymerization. Recombinant (unacetylated) TM2 and N-acetylated striated muscle TM (stTM), long ␣-tropomyosin isoforms with the same N-terminal sequence, different from TM5a, also inhibited depolymerization but were less effective than TM5a. All blocked the pointed end with Tmod1 in the order of effectiveness TM5a >stTM >TM2, showing the importance of the N-terminal sequence and modification. Tmod1-(1-344), lacking the C-terminal 15 residues, did not nucleate polymerization but blocked the pointed end with all three tropomyosin isoforms as does a shorter fragment, Tmod1-(1-92), lacking the C-terminal "capping" domain though higher concentrations were required. An even shorter fragment, Tmod1-(1-48), bound tropomyosin but did not influence actin filament elongation. Tropomyosin-Tmod may function to locally regulate cytoskeletal dynamics in cells by stabilizing actin filaments.

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“…A third approach that uses rules for assignments similar to the ones used by an expert to generate an initial protein fold has been implemented in the program AUTO-STRUCTURE, and applied to protein structure determination. 10,21 The CANDID procedure described here combines features from NOAH and ARIA, such as the use of three-dimensional structure-based filters and ambiguous distance constraints, with the new concepts of network-anchoring and constraintcombination that further enable an efficient and reliable search for the correct fold in the initial cycle of de novo NMR structure determinations.…”

Section: Introductionmentioning

confidence: 99%

Protein NMR Structure Determination with Automated NOE Assignment Using the New Software CANDID and the Torsion Angle Dynamics Algorithm DYANA

Herrmann¹,

Güntert²,

Wüthrich³

2002

Journal of Molecular Biology

1,419

1,079

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Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE crosspeak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE crosspeak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in 0022-2836/02/$ -see front matter q 2002 Elsevier Science Ltd. All rights reserved Present address: P. Gü ntert, RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan.E-mail address of the corresponding author: guentert@gsc.r...

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Solution NMR structure and folding dynamics of the N terminus of a rat non-muscle α-tropomyosin in an engineered chimeric protein 1 1Edited by P. E. Wright

Cited by 64 publications

References 75 publications

Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

Protein NMR Structure Determination with Automated NOE Assignment Using the New Software CANDID and the Torsion Angle Dynamics Algorithm DYANA

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