title: Thymosin beta4 induces a conformational change in actin monomers.
abstract: 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.
authors.affiliation: Muscle Research Unit, Institute for Biomedical Research, University of Sydney, New South Wales, Australia.
authors.forename: Irina V
authors.initials: IV
authors.lastname: Dedova
authors.forename: Olga P
authors.initials: OP
authors.lastname: Nikolaeva
authors.forename: Daniel
authors.initials: D
authors.lastname: Safer
authors.forename: Enrique M
authors.initials: EM
authors.lastname: De La Cruz
authors.forename: Cris G
authors.initials: CG
authors.lastname: dos Remedios
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002152:Calorimetry, Differential Scanning; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006358:Hot Temperature; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011687:Purines / Q000737:chemistry; D011817:Rabbits; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013947:Thymosin / Q000737:chemistry* / Q000378:metabolism; D000099201:Thymosin beta(4)
chemical_list: D020106:Acrylamide; D000199:Actins; D000255:Adenosine Triphosphate; D003545:Cysteine; D008239:Lysine; D009711:Nucleotides; D011687:Purines; D012997:Solvents; D013947:Thymosin; D000099201:Thymosin beta(4); C030985:purine