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Mapping vibrational energy transfer in proteins and investigating its relation to allosteric signal propagation using a non-canonical VET pair

Subject Area Biophysics
Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
Term from 2017 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 349128007
 
Based on simulations a growing number of theoretical studies claim the existence of distinct vibrational energy transfer (VET) pathways, which connect sites of functional importance in proteins. Implications for intramolecular information transfer (allostery) have been discussed. However, experimental evidence for these highly directional pathways is lacking. The appropriate experimental tools need to be developed in order to map out VET in proteins and to scrutinize the existence of the proposed highly directional pathways. Here, we propose a direct experimental investigation of VET pathways in proteins by injecting vibrational energy into a protein with site selectivity and subsequently following its propagation in real-time - which is the experimental counter-part of above mentioned computational approaches. Mapping out VET in proteins requires the ability to site-selectively inject vibrational energy, site-selectively monitor its flow and, importantly, to have the flexibility to chose the respective sites for injection and probing. We aim to achieve this by using our recently developed VET pair of non-canonical amino acids (ncAAs). The pair consists of a VET donor, which is excited by a femtosecond pump pulse and a VET sensor, whose response is measured by a femtosecond infrared probe pulse. The functionality of the VET pair has been demonstrated in our preliminary work on small synthetic peptides. Here, we will expand our preliminary work from simple peptides to proteins. We will use methods for expanding the genetic code in order to achieve precise positioning of VET donor and sensor in the target protein sequences and to obtain the large number and quantities of the required protein mutants by in vivo expression. Donor and sensor will be positioned at various sites both on and off the proposed transfer pathways, which will enable us to compare the experimental energy transfer efficiencies and time scales with the theoretically predicted ones. Detailed maps of vibrational energy transfer in proteins will be obtained. In this way, the experimental challenge posed by the theoretical prediction of distinct VET pathways between allosteric sites can be met. We expect to be able to experimentally proof (or reject) the existence of highly directional VET pathways and their relevance in allosteric signal transmission.
DFG Programme Research Grants
 
 

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