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Papers by Himanshu Pandey
Biochimica et Biophysica Acta (BBA) - General Subjects, 2016
In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transfo... more In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature. We have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques. We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.
Assignment of protein backbone resonances is most routinely carried out using triple resonance th... more Assignment of protein backbone resonances is most routinely carried out using triple resonance three dimensional NMR experiments involving amide 1 H and 15 N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high degree of backbone shift degeneracy. In this backdrop, a novel reduced dimensionality (RD) experiment -(5,3)D-hNCO-CANH-is presented to facilitate (and/or to validate) the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide 15 N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. HiNi and Hi-1Ni-1) for overlapping amide NH peaks. The experiment -encoding 5D spectral information-leads to a conventional 3D spectrum with significantly reduced spectral crowding and complexity. The improvisation is based on the fact that the linear combinations of intra-residue and inter-residue backbone chemical shifts along both the co-evolved indirect dimensions span a wider spectral range and produce better peak dispersion than the individual shifts themselves. Taken together, the experiment -in combination with routine triple resonance 3D NMR experiments involving backbone amide ( 1 H and 15 N) and carbon ( 13 C and 13 C') chemical shifts-will serve as a powerful complementary tool to achieve the nearly complete assignment of protein backbone resonances in a time efficient manner. The performance of the experiment and application of the method have been demonstrated here using a 15.4 kDa size folded protein and a 12 kDa size unfolded protein. 3 Introduction: Over the decades, NMR has emerged as a powerful technique for studying the structure and dynamics of proteins and their complexes in solution. Further, it is the technique of choice for studying conformational properties of intrinsically unstructured proteins (IDPs), and their interactions with their physiological binding partners in solution [1-3]. For various such studies on proteins by NMR, the very first and key requirement is the sequence specific assignment of backbone ( 1 H, 15 N, 13 C' and 13 C') resonances [4,5]. The well-established and most routinely used assignment strategies involve the use of 15 N, 1 H N resolved triple resonance experiments sequentially linking 13 C , 13 C' or 15 N shifts [6-22] and many proteins have been assigned this way (evident from the Biological Magnetic Resonance Bank: http://www.bmrb.wisc.edu). However for proteins exhibiting high degree of backbone amide and carbon shift degeneracy (e.g. -helical proteins or proteins containing disordered fragments including IDPs), getting this information in an unambiguous and time-efficient manner has always remained problematic and challenging. Therefore new or alternative NMR methods and strategies -for rapid and efficient assignment of backbone resonances in such proteins-are required.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2016
In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transfo... more In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature. We have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques. We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.
Assignment of protein backbone resonances is most routinely carried out using triple resonance th... more Assignment of protein backbone resonances is most routinely carried out using triple resonance three dimensional NMR experiments involving amide 1 H and 15 N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high degree of backbone shift degeneracy. In this backdrop, a novel reduced dimensionality (RD) experiment -(5,3)D-hNCO-CANH-is presented to facilitate (and/or to validate) the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide 15 N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. HiNi and Hi-1Ni-1) for overlapping amide NH peaks. The experiment -encoding 5D spectral information-leads to a conventional 3D spectrum with significantly reduced spectral crowding and complexity. The improvisation is based on the fact that the linear combinations of intra-residue and inter-residue backbone chemical shifts along both the co-evolved indirect dimensions span a wider spectral range and produce better peak dispersion than the individual shifts themselves. Taken together, the experiment -in combination with routine triple resonance 3D NMR experiments involving backbone amide ( 1 H and 15 N) and carbon ( 13 C and 13 C') chemical shifts-will serve as a powerful complementary tool to achieve the nearly complete assignment of protein backbone resonances in a time efficient manner. The performance of the experiment and application of the method have been demonstrated here using a 15.4 kDa size folded protein and a 12 kDa size unfolded protein. 3 Introduction: Over the decades, NMR has emerged as a powerful technique for studying the structure and dynamics of proteins and their complexes in solution. Further, it is the technique of choice for studying conformational properties of intrinsically unstructured proteins (IDPs), and their interactions with their physiological binding partners in solution [1-3]. For various such studies on proteins by NMR, the very first and key requirement is the sequence specific assignment of backbone ( 1 H, 15 N, 13 C' and 13 C') resonances [4,5]. The well-established and most routinely used assignment strategies involve the use of 15 N, 1 H N resolved triple resonance experiments sequentially linking 13 C , 13 C' or 15 N shifts [6-22] and many proteins have been assigned this way (evident from the Biological Magnetic Resonance Bank: http://www.bmrb.wisc.edu). However for proteins exhibiting high degree of backbone amide and carbon shift degeneracy (e.g. -helical proteins or proteins containing disordered fragments including IDPs), getting this information in an unambiguous and time-efficient manner has always remained problematic and challenging. Therefore new or alternative NMR methods and strategies -for rapid and efficient assignment of backbone resonances in such proteins-are required.