Development of NMR methodology in solution and solid state
Our focus is to develop new NMR experiments combined with new methods of isotope labeling for structural studies of protein. We have developed several new NMR methods which enable rapid data collection. Some of these methods have been based on the method of G-matrix Fourier transform (GFT) NMR spectroscopy. The pulse sequence parameter for these experiments can be downloaded here.
(i) Secondary structure type assignment, editing and estimation in proteins (J Biomol NMR (2009) 44:185-194)
(ii) Amino acid selective unlabeling (J Biomol NMR (2011) 49:39-51)
(iii) Methyl-edited NOESY: Chemical shift editing of methyl groups in proteins (J Biomol NMR (2010) 48:137-145; J Biomol NMR (2008) 42:149-154)
(iv) Measurement of three-bond scalar coupling constants in proteins (J Biomol NMR (2007) 39:259-263)
(v) Selective identification of specific amino acid residues in NMR spectrum of proteins. (J Biomol NMR (2008) 41: 191-197)
(vi) Measurement of pseudo-contact shifts in paramagnetic proteins (Open Mag Reson J. (2008) 1:16-28)
We have initiated the development of new experimental techniques in solid-state NMR spectroscopy. In order to facilitate rapid data collection, methods such as G-matrix Fourier transform NMR spectroscopy in combination with other fast data acquisition schemes will be employed. In addition, different isotope labeling schemes will be developed and tested. These experiments will be applied to different biological systems such as membrane proteins and amyloid fibrils.
Structural studies of IGF-system
Our research work focuses on studying important biological systems from structural and functional aspects using NMR spectroscopy. In collaboration with Prof. Steven Rosenzweig's group in UMC, South Carolina, USA, we are involved in the Structural and functional studies of Insulin-like growth factor binding proteins. In recent years, the Insulin-like growth factor (IGF) system has become the focus of clinically important targets of cancer therapeutics. This is due to the fact that the IGF system plays an important role in the growth and function of the human body. This system comprises the following components: (i) Two peptide hormones, IGF-1 and -2, (ii) type 1 and type 2 IGF receptors, (iii) six IGF-binding proteins (IGFBP; numbered 1-6) and (iv) IGFBP proteases.
IGF-1 and -2 are small signalling peptides (~7.5 kDa) that play central roles in cell growth, proliferation, differentiation and metastasis. They stimulate action by binding to specific cell surface receptors (IGF-1R) evoking subsequent response inside the cell. The activity of the IGFs is regulated by six soluble IGF binding proteins (IGFBP) which range in 22-31 kDa in size and share overall sequence and structural homology with each other. IGFBPs bind strongly to IGFs (KD ~ 300-700 pM) to ensure that all the circulating IGF in the blood stream is sequestered and inhibit the action of IGFs by blocking their access to the receptors. Proteolysis of the IGFBPs dissociates IGFs from the complex, enabling them to bind and activate the cell surface receptors. Our research focuses on mapping these interactions at the structural level using NMR spectroscopy.
1. Swain et al. Arch. Biochem Biophys (2010) 501: 195-200.
2. Rosenzweig and Atreya Biochem Pharmacol (2010) 80: 1115-1124.
We recently discovered that the C-terminal fragment of hIGFBP-2 (residues 249-289) self-assembles spontaneously and reversibly into nanotubular structures under non-reducing conditions and remains as a monomer under reducing condition. These nanotubular structures were studied extensively by transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD). This is the first reported nanotubular structures formed spontaneously by polypeptides using covalent disulphide linkages We are now pursuing this work further to understand the molecular mechanisms under which such nanotubular structures form and use this to develop IGFBP-based therapeutics for targeting tumors. Due to the presence of a RGD motif in this fragment, it has applications in targeting integrin tumors. A complete biophysical and biochemical study of the formation of the tubular structures is in progress.
Swain et al. ChemComm (2010) 46: 216-218.
We have developed new computational methods for improvement of quality of one and two dimensional NMR spectra. This is useful for a number of different applications such as resonance assignments, accurate measurement of NMR parameters, automated peak picking and line-shape fitting in spectra. The method works by applying a set of intensity transforms to each point in the spectrum. Some of these methods are non-linear and derived from methods used in digital image processing. A significant improvement in the quality of the spectrum can be achieved while preserving the quantitative aspects of the spectra. To facilitate easy spectral handling and processing, a graphical user interface (GUI) has been designed which takes as input the NMR spectrum and applies the desired transforms. The software will be put soon on this website.