Areas of Research
A new strategy for monitoring small motions by FRET was conceived and implemented by using two small fluorescent amino acids that fit into the protein structure without distortion. Protein motion changes the dipole orientation between the probes, resulting in a significant change in FRET even in the absence of any large distance change.
A sCD4 protein was prepared in a cell-free translation system. It is going to be introduced a series of fluorescent amino acids at desired position. The fluorescent sCD4 protein mimics will change color while it binds to the HIV gp120 protein. Thus, it will be developed into a probe to diagnose HIV infection at an early stage to help prevent the spread of the epidemic.
Two pyrenylalanine analogs have been incorporated into dihydrofolate reductase (DHFR) at positions 16 and 49 to obtain an excimer formation, which is more sensitive to small changes in distance than FRET pairs (a constraint imposed by the dimensions of the enzyme). The excimer formation was used to study the dynamics of DHFR under both single and multiple turnover conditions. More fluorescent amino acids will be synthesized and incorporated into different positions of DHFR to study its function and dynamics in the near future.
Thiothreonine and its analogs were synthesized and incorporated into DHFR at predetermined position, and the elaborated proteins were modified site-specifically at the thiothreonine residue with a fluorophore. Aspartic acid derivatives also were synthesized and incorporated into DHFR at desired position to study the function of enzyme.
A p-thiophenylalanine was synthesized and incorporated into the active site (position 274) of vaccinia DNA topoisomerase IB by in vitro translation. The modification, which resulted in replacement of the nucleophilic tyrosine OH group with SH, retained DNA topoisomerase activity and did not alter the DNA cleavage site. However, the modified topoisomerase effected relaxation of supercoiled plasmid DNA at a rate about 16-fold slower than the wild-type enzyme. The thiophenylalanine-induced DNA cleavage rate was 30 times lower than for the wild-type enzyme. The changed rates were due to alterations in the bond strengths of the obligatory intermediates.
- Chen,Shengxi * . Novel materials to increase pleasure and enhance erection (ASUF 30006147). ASU FDN (5/1/2014 - 10/31/2015).
- Chen,Shengxi * . Fluorescent protein sensor to diagnose HIV at low cost. GATES (BILL & MELINDA) FDN (5/1/2012 - 4/30/2014).
* principal investigator