Ca²⁺-binding and spectral properties of the common region of surface exposed Lig proteins of Leptospira

Abstract

Pathogenic Leptospira protein LigA and LigB are conserved at the N-terminal sequence. In our earlier report, we have presented the spectral properties of individual Big domain of Lig proteins, and showed that an individual domain binds Ca²⁺. Here we demonstrate that apart from Ca²⁺-binding properties, the spectral properties (such as doublet Trp fluorescence) shown by an individual domain are almost retained in the protein with many such domains (which could be easily be called as a multimer of an individual tandem repeat). Presence of Asp and Asn in a stretch of sequence in all tandem repeats points towards the possibility of their involvement in Ca²⁺-binding. 

Acknowledgements

This work was supported in part by the Biotechnology Research and Development Corporation (BRDC) and DBT as well as DST (Govt. of India). Rajeev Raman is supported by a senior research fellowship from the Council of Scientific and Industrial Research, Government of India.

Figures and Tables

Figure 1 Tb³⁺ binding to LigCon. 10 µM of purified recombinant LigCon was suspended in 50 mM Tris (pH 7.2) and 50 mM KCl and excited at 285 nm. Emission spectrum was recorded from 300 nm to 580 nm. Aliquots of terbium chloride (0 to 2 mM) were added in the protein solution and spectra were recorded. The intensity at 545 nm was plotted against terbium chloride concentrations. Inset shows emission spectra between 450 and 560 nm upon terbium chloride addition. The two peaks at 485 and 545 nm are seen.

Figure 2 (A) Far-UV CD spectra of LigCon. CD Spectra were recorded using protein concentration of 1.35 mg/ml in a buffer containing 15 mM Tris (pH 7.5), 100 mM KCl and 1 mM DTT. Final calcium concentrations were 0, 100, 200 and 500 µM. Direction of arrows follows the increasing order of calcium concentration. (B) Near-UV CD spectra of LigCon. Protein concentration at 0.86 mg/ml in buffer containing 50 mM Tris (pH 7.5) and 100 mM NaCl was used in a 1 cm path length cuvette. Calcium chloride was added to a final concentration of 0, 100 and 500 µM. Steady-state fluorescence spectra of LigCon and (C) effect of Ca²⁺ and (D) Mg²⁺. 10 µM of protein in 20 mM Tris (pH 7.5), 150 mM KCl and 1 mM DTT was excited at 295 nm. Aliquots of calcium chloride or magnesium chloride from respective stock solutions were added until saturation was reached. The figure shows Trp fluorescence in the presence of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1 and 2 mM of CaCl₂.

Figure 3 Sequence of the common region (LigCon) indicating a consensus sequence in each tandem repeat (shown in bold and underlined).

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