Abstract
The signal recognition particle (SRP) play a vital role in the co-translational targeting of proteins across the membrane. The SRP present in chloroplast exist in two pools, one containing the 54 kDa domain (cpSRP54) and the other containing a 43 kDa domain (cpSRP43). The cpSRP43 is involved in the targeting of the light harvesting chlorophyll-binding protein (LHCP) to the thylakoids. cpSRP43 as such contains 308 amino acids and hence it is technically challenging to solve the three dimensional structure by NMR. Domain structure of cpSRP43 reveals that it contains three chromo domains and one Ankyrin repeat domain. In this study, various chromo domains are cloned separately into E.coli. expression vectors and purified by affinity chromatography. Stability and biophysical properties of all the chromo domains are characterized. Structural characterizations of all the chromo domains were done by NMR. Three-dimensional structure of Chromo3 domain is investigated in detail. The resonance assignment of the Chromo3 domain is accomplished by homonuclear and heteronuclear NMR spectroscopy. Three dimensional structure calculation of Chromo3 domain is done using ARIA / CNS. Functional properties of all individual domain of cpSRP43 were also characterized. Ability of various domains to bind towards the light harvesting chlorophyll-binding protein was studied in detail. The heat of binding of the L18 peptide to various domains of cpSRP43 was measured using isothermal titration calorimetry (ITC), and the binding isotherms with various domains were constructed. Binding of the L18 peptide to cpSRP43 is also investigated in detail using fluorescence spectroscopy. Experiments were done by monitoring the changes of the intrinsic fluorescence of the individual domains upon binding to the L18 peptide. Changes in spectroscopic signal were monitored both in steady state and in stop flow fluorescence spectrometer. Changes in the fluorescence signal of the L18 peptide upon binding to individual domains was also monitored with 5-carboxy fluorescein tagged L18 peptide. NMR analysis for the binding was also done. Chemical shift perturbation analysis revealed that the binding site for the L18 peptide is maximally located at the Ankyrin repeat domain of the cpSRP43. Critical analysis of the NMR data for the titration of various domains of cpSRP43 with L18 peptide in terms of line broadening, disappearance of cross peaks, evolution of new cross peaks etc., also confirms that the Ankyrin repeat domain of cpSRP43 binds to the L18 peptide. The dissociation constant for the binding has also been calculated.