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Daniel Safer

Publications and source records attributed to Daniel Safer.

6 recordsLinked to original sources
PubMed2005

Thymosin beta4 induces a conformational change in actin monomers.

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin beta(4) (tbeta(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tbeta(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 A and decreases the distance between the purine base of bound ATP (epsilonATP) and Lys-61 by 1.9 A, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tbeta(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tbeta(4) binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tbeta(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tbeta(4) and cofilin compete for actin binding. Tbeta(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tbeta(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tbeta(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding.

Acrylamide↗
PubMed2005

Molecular extensibility of mini-dystrophins and a dystrophin rod construct.

Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophin's large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Delta2331 consists of repeats (R) and hinges (H) H1-R1-2 approximately H3 approximately R22-24-H4 linked by native (-) and non-native (approximately) sequence. This is compared to its core fragment R2 approximately H3 approximately R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23-42 degrees C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied betaI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.

Algorithms↗
PubMed2002

Kinetic characterization of the weak binding states of myosin V.

Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K(d) = 4 microM in the presence of ATP gamma S at 50 mM KCl, which is 10-20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K(d) = 0.2 and 2 microM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.

Adenosine Triphosphate↗
PubMed2002

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments.

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.

Animals↗
PubMed2002

Thymosin beta4 regulation, expression and function in aortic valve interstitial cells.

BACKGROUND AND AIMS OF THE STUDY: Previous research has demonstrated that tenascin-C, an extracellular matrix protein involved in bone development and mineralization, was specifically present in calcified aortic valves, always in association with matrix metalloproteinase (MMP)-2, and was not detectable in non-calcified human aortic valves. The aim of the present study was to identify downstream targets of tenascin-C in aortic valve interstitial cells. METHODS: Subtractive hybridization was performed using sheep aortic valve interstitial cells (SAVIC) grown on a substrate of collagen plus tenascin-C, versus cells grown on type I collagen alone. RESULTS: Subtractive hybridizations revealed that nearly 70% of the clones isolated contained the sequence for thymosin beta-4, an actin-binding protein, also associated with mineralization, regulation of MMPs and inflammation. In cell culture studies, it was shown that both thymosin beta4 and thymosin beta4 sulfoxide are produced by SAVIC. When aortic valve interstitial cells were grown on a substrate of tenascin-C, thymosin beta4 expression was up-regulated. The addition of thymosin beta4 to aortic valve interstitial cell cultures resulted in a re-orientation of cytoskeletal F-actin, and induction of MMP-2. However, thymosin beta4 antisense oligonucleotide transfection did not suppress MMP-2 expression. CONCLUSION: Thymosin beta4 is up-regulated by tenascin-C, and may be involved in the primary initiation of valvular calcification.

Adult↗