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CheY/ Design Proteins

CheY Design Proteins
Different exchange rates of amino acids (colored according to their exchange rate constants) within Di_III-14 ranging from seconds to days in Hydrogen-Deuterium exchange experiment monitored by NMR reveal the rough energy surface of this protein. 

In order to improve the function of the designed proteins over the naturally occurring proteins, protein engineers opt for random sequences which are hardly or never observed in naturally occurring proteins from the amino acid sequence space, bring the complex consequences of these sequences which do not have evolutionary filter on the folding free energy landscape. In thermodynamic studies, the folding free energy surface of designed protein appears apparently smooth in thermal and chemical denaturation. The kinetic signatures from the unusual sequence are still elusive. Here we try to emphasize the effect of the kinetically populated intermediates which make the energy surface rough, arise from the complexity of sequence in the designed protein. To understand the complex relationship between these sequences, structure, function in the context of folding free energy landscape, we started working with a designed protein called Di-III_14, with βα topology from David Baker's Lab. Koga et al. (Nature 2012) showed that this protein showed simple two state transition in chemical denaturation experiment with a modest stability and this protein is stable at higher temperature also. With the help of wide range of biophysical tools like Circular dichroism (CD), stopped flow fluorescence and NMR, a very complex energy landscape is revealed in water with several kinetic intermediates between folded and unfolded state of this protein. Actually, the sequence complexity within the protein gives rise of several electrostatic and hydrophobic networks within it and the contribution of these networks makes the protein interior more packed, comparable to the hyper thermophilic protein of its structural orthologues. Thus the unbiased exploration of these kind of unusual sequences from the sequence space extemporizes our understanding about the "central dogma "of protein folding, sequence –structure – function relationship. In future, this kind of work will help the protein engineers to optimize the thermodynamic as well as kinetic consequences coming from sequences on the folding free energy landscape which is needed to be smooth for the more improved functions.