Figure 1.
The sequence of (a)TAR RNA and (b) Tat peptide along with different lysine modifications and their positions.
Figure 2.
CD spectra TAR RNA and the effect of binding of different peptides on the structure of the RNA at 25 °C.
(□) 2 μM RNA, (○) 10 μM Peptide. All CD spectra were collected in a buffer containing 10 mM sodium cacodylate, 70 mM NaCl and 0.l mM EDTA at pH 7.5.
Figure 3.
Thermal melting profile of TAR RNA alone and in the presence of different molar ratio of the peptides.
(□) 1:0 molar ratio RNA:peptide (○)1:1 molar ratio RNA:peptide (△)1:2 molar ratio RNA:peptide (▽)1:3 molar ratio RNA:peptide. All the melting profiles were collected at 260 nm wavelength. The buffer condition were same as described in Figure 2 caption.
Figure 4.
ITC titration profile of TAR RNA with Tat and different modified peptides at 25 °C.
Upper panel shows the baseline corrected experimental data for peptide binding, lower panel shows the molar heats of binding (□) plotted against the peptide to RNA molar ratio. Buffer condition was as described in the caption to Figure 2. Molar heat of binding is calculated by integration of the area under the curve of each heat burst using the origin version 7.0 software (Microcal, Inc.; Northampton, MA). Fitting of ITC data (shown as solid line) was done using model for two set of binding [38] given in origin version 7.0 software (Microcal, Inc.; Northampton, MA).
Figure 5.
SPR sensogram showing binding kinetics of Tat peptide along with the fitting curves obtained from fittings using two independent site model.
The concentration series of peptides used for studying kinetics range from 9.5 nM to 1.2 μM. The expepriments were conducted in the 10 mM sodium cacodylate buffer containing 70 mM NaCl and 0.l mM EDTA at pH 7.5. The representative fitting curves of sensogram for Tat peptide binding is shown in red color.