Christopher Marai

Experimental work on by our collaborators has revealed tremendous structural information about the native state and folding NTL9.[1-4] In spite of this significant experimental progress, atomic level details of the unfolding path remain unknown and have yet to be addressed by computational investigation. Due to its compact size and excellent experimental characterization it is an ideal system to study computationally.

Current efforts aim to sample the folding pathway of NTL9 with high temperature all-atom MD simulations in implicit solvent. This method has been used successfully in the literature to study the folding and transition state ensembles of other similar sized proteins.[6] We investigated the unfolding path of NTL9 by unfolding the protein independently 100 times for 500ps using all-atom MD implicit solvent. Unfolding was induced by 350K and 400K temperatures. The free energy profile for a series of reactions coordinates was determined from data of 100 unfolding runs and indicates NTL9 folds in a two-state manner. This result is consistent with known experimental evidence of NTL9 folding.[1-4] Moreover, our preliminary calculations indicate specific non-native interactions in the denatured state as suggested by experiment.[7] The current methods were implemented on 10, 500Mhz nodes on the serial node cluster in the department of chemistry (called RAM). Progress has since been limited by CPU time and additional CPU time will be required to uncover some of the questions in our aims as listed below.

What is the 3D structure of the transition state (TS) of unfolding?
Refolding and mutational experiments have revealed macroscopic details of the transition state ensemble (TSE) of NTL9. In the past such results coupled with molecular dynamics simulations have been used to determine an atomic level description of the transition state ensemble of other proteins.[6] Currently, the experimentally dependent Phi-RMSD method[6] has been implemented in discovering TSE structures.
Should the denatured state be included in calculation of phi-values?
On going experimental work by the Raleigh group has highlighted that conventional phi-value analysis maybe limited in its effort to identify the importance of residues on the folding pathway. Specifically, current calculation of phi-values requires mutational analysis of amino acids and considers changes between the native state and the TS.

ABSTRACT