1
THE EFFECTS OF MOUSE TWINFILIN-1 ON THE STRUCTURE AND DYNAMICS OF ACTIN
Veronika Kollár1, Roland Kardos1, Pekka Lappalainen2, Miklós Nyitrai 1and Gábor Hild1
1 University of Pécs, Medical School, Department of Biophysics, 7624 Pécs, Szigeti street 12, Hungary
2Program in Cellular Biotechnology, Institute of Biotechnology, PO Box 56, 00014 University of Helsinki, Finland
Introduction Materials and methods
Acetone-dried muscle powder was obtained from rabbit skeletal muscle as was described earlier by Feuer and
The actin cytoskeleton of eukaryotic cells plays a key role in many processes. The structure and dynamics of the cytoskeleton are regulated by a large number of proteins that interact with monomeric and/or filamentous actins.
Several actin binding proteins can directly regulate the elongation and nucleation of actin filaments. Twinfilin is a 37-40 kDa protein composed of two ADF-homologue domains connected by a short linker. Twinfilins are actin monomer binding/sequestering proteins which play a crucial role in the polymerization/depolymerization cycle of actin filaments. In our work we studied the effects of the mouse twinfilin 1 (TWF1) on the monomeric actin.
Acetone-dried muscle powder was obtained from rabbit skeletal muscle as was described earlier by Feuer and his colleagues (Feuer, 1948). The G-actin was prepared according to the method of Spudich and Watt (Spudich, 1971).
The mouse twinfilin 1 was expressed inEscherichia coliBL21 (DE3) pLysS expression system. Horiba Jobin Yvon (Longjumeau, France) Fluorolog-3 and Perkin-Elmer (Waltham, MA) LS50 spectrofluorometer were used for the fluorescence measurements.
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i SV SV
i Q K Q K
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The affinity of the twinfilin for actin was determined by using the following equation:
Fluorescence quenching experiments:
The fluorescence quenching experiments were analyzed with the following equation:
Differential Scanning Calorimetry (DSC) experiments:
DSC measurements were completed on a Setaram MicroDSCIII calorimeter with a heating rate of 0.3 K/min.
Nucleotide Exchange Assay:
The nucleotide exchange assay was completed onε-ATP actin monomers in the absence and presence of TWF1 with an Applied Photophysics stopped-flow equipment.
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Results
Fluorescence anisotropy measurements Stopped flow experiments Protein expression
His-tagged twinfilin-1 kDa
116 66.2 45 35
25
DSC measurements Fluorescence quenching experiments
The change of fluorescence anisotropy in the presence of increasing twinfilin-1 concentration.
Figure 2.
Kinetics of nucleotide exchange on the ε-ATP labelled G-actin (1 μM).
Figure 3.
Figure 1.
SDS - polyacrylamide electrophoretogram of the His - tagged mouse twinfilin 1.
18.4 14.4
o The affinity of twinfilin for the actin monomer was determined by fluorescence anisotropy (KD=0.07±0.02 µM) (Figure 2.) Figure 5.
The DSC curve of 23 μM Ca-G-actin in the presence of 23 μM twinfilin.
Figure 4B.
The Stern-Volmer plot for ε-ATP labelled monomeric actin (5 μM) in the presence of twinfilin (15 μM).
The Stern-Volmer plot for ε-ATP labelled monomeric actin (5 μM) in the absence of twinfilin.
Figure 4A.
•
The decreased nucleotide exchange rate and the decreased K value indicate that the nucleotide binding cleft is shifted to a closedConclusions
D
o The nucleotide exchange assay showed that the exchange rate decreased from 0.012 s-1to 0.003 s-1in the presence of twinfilin (Figure 3.).
o The Steady-state fluorescence quenching experiments showed that the KSVfor the actin bound ε-ATP decreased from 0.25 M-1to 1.2∙10-15M-1in the presence of twinfilin (Figure 4.).
o In the DSC experiments the melting temperature of twinfilin bound actin (Tm= 59°C) was higher compared to the twinfilin free actin monomers (Tm= 56.1°C) (Figure 5.).
•
The decreased nucleotide exchange rate and the decreased KSVvalue indicate that the nucleotide binding cleft is shifted to a closed conformationalstatein the presence of twinfilin.•
The higher Tmvalue for the twinfilin bound actin monomers indicates that the conformational change of the nucleotide binding cleft is accompanied by theincreased thermodynamic stabilityof the actin monomers.The present work was supported by TÁMOP-4.2.1.B-10/2/KONV-2010-0002 "A Dél-Dunántúli Régió egyetemi versenyképességének fejlesztése" and TÁMOP-4.2.2-08/1-2008-0011, "SP! IKT - Science, Please! Innovatív Kutatói Team" and TÁMOP-4.2.2. B-10/1-2010-0029 "Tudományos képzés műhelyeinek támogatása a Pécsi Tudományegyetemen" and “Baross Gábor Programme – South Danubian region: REG-DD-09-1-2009-0009 Tirfm_09”and grants from the Hungarian Scientific Research Fund (OTKA grant K77840 to Miklós Nyitrai and OTKA-PD83648 to Beáta Bugyi).
Miklós Nyitrai holds a Wellcome Trust International Senior Research Fellowship in Biomedical Sciences. DEPARTMENT OF
BIOPHYSICS PÉCS, SZIGETI STREET 12. H-7624 PHONE: (36) 72 536260 FAX: (36) 72 536261 UNIVERSITY OF PÉCS MEDICAL SCHOOL
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