Technology Review - Structure Determination of Proteins in Solution by Nuclear Magnetic Resonance Spectroscopy

Each cell in the human body consists of a myriad of biological macromolecules and organelles, which are required for various cellular functions and metabolism. A key component in such a system comprises of proteins, which play a crucial role in proper functioning of the cell. There are an estimated 100,000 different proteins present in the human body.1 The activity of a protein molecule inside the cell is indirectly governed by the overall fold of the individual polypeptide chains, or in other words, their three dimensional (3D) structures in space.2 Thus, the knowledge of the 3D structure of a given protein is most essential for a complete understanding of its function inside the cell. Many diseases in humans such as Alzheimers', Parkinsons', Prion disease, Cystic fibrosis, cancers etc. are attributed as the result of malfunctioning of proteins.1 Further, knowledge of the 3D structure of a protein, involved in a disease, is eventually used in designing its target drugs. Such a sphere of activity is popularly called as quantitative structure activity relationship (QSAR).

Mechanism of Initiation, Association, and Formation of Amyloid Fibrils Modeled with the N-Terminal Peptide Fragment, IKYLEFIS, of Myoglobin G-Helix

Some peptides and proteins undergo self-aggregation under certain conditions, leading to amyloid fibrils formation, which is related to many disease conditions. It is important to understand such amyloid fibrils formation to provide mechanistic detail that governs the process. A predominantly alpha-helical myoglobin has been reported recently to readily form amyloid fibrils at a higher temperature, similar to its G-helix segment. Here, we have investigated the mechanism of amyloid fibrils formation by performing multiple long molecular dynamics simulations (27 mu s) on the N-terminal segment of the G-helix of myoglobin. These simulations resulted in the formation of a single-layered tetrameric beta-sheet with mixed parallel and antiparallel beta-strands and this is the most common event irrespective of many different starting structures. Formation of the single-layered tetrameric beta-sheet takes place following three distinctive pathways. The process of fibril initiation is dependent on temperature. Further, this study provides mechanistic insights into the formation of multilayered fibrilar structure, which could be applicable to a wider variety of peptides or proteins to understand the amyloidogenesis

Structure prediction of a multi-domain EF-hand Ca2+ binding protein by PROPAINOR

PROPAINOR is a new algorithm developed for ab initio prediction of the 3D structures of proteins using knowledge-based nonparametric multivariate statistical methods. This algorithm is found to be most efficient in terms of computational simplicity and prediction accuracy for single-domain proteins as compared to other ab initio methods. In this paper, we have used the algorithm for the atomic structure prediction of a multi-domain (two-domain) calcium-binding protein, whose solution structure has been deposited in the PDB recently (PDB ID: 1JFK). We have studied the sensitivity of the predicted structure to NMR distance restraints with their incorporation as an additional input. Further, we have compared the predicted structures in both these cases with the NMR derived solution structure reported earlier. We have also validated the refined structure for proper stereochemistry and favorable packing environment with good results and elucidated the role of the central linker

Chemical shift based editing of CH3 groups in fractionally 13C^{13}C-labelled proteins using GFT (3,2)D CT-HCCH-COSY: stereospecific assignments of CH3CH_3 groups of Val and Leu residues

We propose a (3,2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing $^13$C-$1^H$ cross peaks of $CH_3$ groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the $^{13}C$-$^1H$ cross peaks of $CH_3$ groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) $^{13}C$-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) $^{13}C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins