PDBsum entry 1vii

Actin binding

PDB id: 1vii

Contents

Protein chain

36 a.a.

References listed in PDB file

Key reference

Title

Nmr structure of the 35-Residue villin headpiece subdomain.

Authors

C.J.Mcknight, P.T.Matsudaira, P.S.Kim.

Ref.

Nat Struct Biol, 1997, 4, 180-184.

PubMed id

9164455

Abstract

The NMR structure of an autonomously folding subdomain from villin headpiece is reported. It forms a novel three helix structure with the actin-binding residues arrayed on the C-terminal helix.

Secondary reference #1

Title

A thermostable 35-Residue subdomain within villin headpiece.

Authors

C.J.Mcknight, D.S.Doering, P.T.Matsudaira, P.S.Kim.

Ref.

J Mol Biol, 1996, 260, 126-134. [DOI no: 10.1006/jmbi.1996.0387]

PubMed id

8764395

Figure 1.
Figure 1. Generation of C-terminal peptides by trypsinolysis of headpiece. Trypsinolysis was carried out in 1 M GuHCl. After quenching at the specified times by addition of acetic acid to 5% (v/v), digestion products were analyzed by reverse-phase high pressure liquid chromatography (HPLC). The data are plotted as a percentage of the total absorbance of all peaks at 229 nm. The 56 to 65 fragment is the complete digestion product. Filled triangles, 32 to 76; filled circles, 38 to 76; open triangles, 56 to 65; open circles, 56 to 73; open squares, 56 to 71; open diamonds, 56 to 70. Methods: Headpiece was expressed from the plasmid pDDHP10 (Doering, 1992) in E. coli using a phage T7 expression system (Studier et al., 1990). BL21(DE3)pLysS cells (Novagen) transformed with the desired plasmid were grown in LB media containing ampicillin at 100 mg ml -1 . Expression was induced by addition of isopropyl b-D-thiogalactopyranoside (120 mg ml -1 final concentration) at an optical density between 0.5 and 0.8 at 590 nm. Cells were harvested by centrifugation two to three hours after induction. Cell pellets from one liter bacterial cultures were brought up in 25 ml of buffer A (3 M urea, 200 mM NaCl, 0.1 mM EDTA, 50 mM Tris (pH 8.0)) and sonicated with a probe sonicator for five minutes to lyse the cells. The samples were then centrifuged at 180,000 g for 30 minutes and the supernatants were applied to an ACA34 size exclusion column (1000 cm × 5 cm, Sepracor) running in buffer A. Fractions were collected and assayed by sodium dodecyl sulfate/gel electrophoresis (Scha˜gger & von Jagow, 1987). Fractions containing the desired peptides were pooled, dialyzed against water and then concen- trated by lyophilization. The crude peptide was solublized in 5% acetic acid and purified by reverse-phase HPLC on a C18 column (Vydac, 2 cm × 25 cm) eluted with a water/acetonitrile gradient containing 0.1% (v/v) trifluoroacetic acid (TFA). Peptide identity was confirmed by laser desorption mass spectrometry (Finnigan MAT). Peptide concentrations were determined by UV absorbance at 280 nm (Edelhoch, 1967). Trypsin digestion was carried out with chymotrypsin- free bovine trypsin. Bovine trypsin (Sigma) was solvated to 5 mg ml -1 in 100 mM Tris, 20 mM CaCl2 (pH 7.5), and 10 ml was loaded on to 5 cm × 2 cm soybean trypsin inhibitor-linked agarose column (Pharmacia). The column was washed with 20 ml of running buffer containing tryptamine (2.5 mg ml -1 ) to elute any contami- nating chymotrypsin. The purified trypsin was eluted with 3 mM HCl, 20 mM CaCl2, and stored in aliquots frozen at -20°C. The digestion reaction contained 100 mM headpiece in 1 M GuHCl, 100 mM ammonium bicarbonate (pH 09). Trypsin was added to a final concentration of 3 mM.

Figure 2.
Figure 2. CD measurements of headpiece and C-terminal peptides. a, Thermal unfolding monitored by CD. For ease of comparison, the data have been normalized to fraction unfolded after subtraction of the folded and unfolded baseli- nes. Protein concentrations were 20 mM in 10 mM phosphate (pH 7.0), with 150 mM NaCl and 500 mM EDTA. b, CD spectra of headpiece and headpiece fragments at 25°C. Protein concentrations were 20 mM in 10 mM phosphate (pH 7.0). c, Guanidine hydrochloride denatura- tion of HP-36 in H2O. The samples were 20 mM HP-36 in 150 mM NaCl, 25 mM acetate (pH 5.0), at 4°C. The line is the best fit to the equation described by Santoro & Bolen (1988). For this fit DG = 3.3 kcal mol -1 , and the m value was 800 cal mol -1 M -1 . Insert shows the linear extrapolation of the DG values de- termined in the transition region to 0 M GuHCl. Methods: The expression plasmid for HP-45, pVHP32-76, was con- structed by PCR from the parent headpiece expression plasmid pDDHP10 (Doering, 1992). Ex- pressed HP-35 retains the additional N-terminal initiator methionine residue, so the resultant peptide is 36 residues long and is referred to as HP-36. An initial expression plas- mid for HP-36, pVHP42-76, was constructed by single-stranded mu- tagenesis of pVHP32-76, deleting the first ten codons after the initi- ator methionine codon. This plas- mid expressed poorly, so the coding region was recloned into the pDDHP10 plasmid backbone (pVHP42-76b). The pVHP42-76b plasmid was found to express the HP-36 fragment at levels sufficient for purification. Recombinant DNA techniques were based on standard protocols (Sambrook et al., 1989). Constructs were confirmed by DNA sequencing (Sanger et al., 1977). HP-45 and HP-36 were expressed and purified as described in the legend to Figure 1, except that buffer A was replaced by 50 mM phosphate buffer (pH 7.0), and the ACA34 column was replaced by a Sephadex G-50 column (1000 × 2.5 cm) run in 50 mM phosphate. The synthetic peptide HP-35 was synthesized using standard solid phase FMOC peptide synthesis protocols on an Applied Biosystems model 430A automated peptide synthesizer. The peptide was cleaved from the resin with TFA and purified by HPLC as described in the legend to Figure 1. CD spectra were recorded on an AVIV 60DS or 62DS spectrometer equipped with a thermoelectric sample temperature controller. Samples for wavelength spectra contained 20 mM peptide in 10 mM phosphate buffer (pH 7.0). The cell path length was 0.1 cm. The spectra were collected as the average of six scans, using a three-second integration time at 1.0 nm wavelength increments. Spectra were baseline-corrected against the cuvet with buffer alone. Samples for the thermal unfolding experiments were 20 mM peptide in 150 mM NaCl, 500 mM EDTA, 10 mM phosphate buffer (pH 7.0). A 1.0 cm path-length cell was used with continuous stirring. Data points were collected at one-degree intervals with an integration time of 20 seconds after a one-minute equilibration period at the desired temperature. The thermal unfolding curves were baseline-corrected by subtraction of a buffer only control experiment. In all cases the thermal unfolding was over 95% reversible and the Tm values did not change after repeated thermal

The above figures are reproduced from the cited reference with permission from Elsevier