Long-Range Contacts in Unfolding of Two-State Proteins.

Predicting the unfolding rates of proteins remains complicated due to the intricacy present in the unfolding pathway of proteins and further it was observed that the experimental unfolding data were less while compared to folding kinetics. The aim of our present work is to show the variation in long-range contacts observed in various sequence separation bins belonging to all-α, all-ß and mixed structural classes of 52 two-state proteins. In this work linear regression technique have been used and regression equations were developed using long-range contacts observed from various sequence separation bins. Also nine topological parameters developed from the 3-D structures of proteins are related with their experimental unfolding rates and their variation in correlation coefficient is observed before and after structural classification. The present work aims to show that long-range contacts formed between residues which are sequentially far and spatially close in the 3-D structure of proteins play a crucial role in the unfolding mechanism of proteins. Also importance of long-range contacts in various experimental and theoretical studies of protein folding along with NMR studies of the unfolded non-native states of proteins have been discussed.

Preferential water exclusion in protein unfolding.

Association of water with protein plays a central role in the latter's folding, structure acquisition, ligand binding, catalytic reactivity, oligomerization, and crystallization. Because these phenomena are also influenced by the net charge content on the protein, the present study examines the association of water with cytochrome c held at different pH values so as to allow its side chains to ionize to variable extents. Equilibrium unfolding of differently charged cytochrome c molecules in water-methanol binary mixtures, where the alcohol acts as the cosolvent denaturant, was used to quantify the preferential exclusion of water during the unfolding transition. The extent of exclusion was found to be related to the net-charge-dependent molecular expansion of the protein in an alcohol-free aqueous medium. The degree of water exclusion was also found to be linearly related to the observed rate of protein unfolding, where the net charge contents of the initial and final states are the same. The results suggest that side-chain ionization, molecular expansion due to charge repulsion, and hence the loss of tertiary contacts lead to additional water-protein association. Protein unfolding rates appear to be linearly correlated with the effective number of water molecules excluded across the end states of unfolding equilibria.

Sequence and structural analysis of two designed proteins with 88% identity adopting different folds.

Protein folding is a natural phenomenon by which a sequence of amino acids folds into a unique functional three-dimensional structure. Although the sequence code that governs folding remains a mystery, one can identify key inter-residue contacts responsible for a given topology. In nature, there are many pairs of proteins of a given length that share little or no sequence identity. Similarly, there are many proteins that share a common topology but lack significant evidence of homology. In order to tackle this problem, protein engineering studies have been used to determine the minimal number of amino acid residues that codes for a particular fold. In recent years, the coupling of theoretical models and experiments in the study of protein folding has resulted in providing some fruitful clues. He et al. have designed two proteins with 88% sequence identity, which adopt different folds and functions. In this work, we have systematically analysed these two proteins by performing pentapeptide search, secondary structure predictions, variation in inter-residue interactions and residue-residue pair preferences, surrounding hydrophobicity computations, conformational switching and energy computations. We conclude that the local secondary structural preference of the two designed proteins at the Nand C-terminal ends to adopt either coil or strand conformation may be a crucial factor in adopting the different folds. Early on during the process of folding, both proteins may choose different energetically favourable pathways to attain the different folds.