Triplet state spectroscopy reveals involvement of the buried tryptophan residue 310 in Glyceraldehyde-3-phosphate dehydrogenase (GAPD) in the interaction with acrylamide.

Using conventional steady state and time resolved fluorescence study of the interaction between a multi-tryptophan protein and a quencher, it is difficult, if not impossible to identify the particular tryptophan residue/residues involved in the interaction. In this work we have exemplified the above contention using a multi-tryptophan protein, Glyceraldehyde-3-phosphate dehydrogenase (GAPD) from rabbit muscle having three tryptophan (Trp) residues at positions 84, 193 and 310 and a neutral quencher acrylamide in Tris buffer of pH 7.5. From the steady state and time resolved fluorescence quenching (at 298 K) with acrylamide K, K and k for the system have been calculated. Low temperature phosphorescence (LTP) spectra at 77 K of GAPD in suitable cryosolvent is known to exhibit two (0,0) bands corresponding to two tryptophan residues 193 and 310. Using the LTP study of free GAPD and GAPD - acrylamide it is possible to identify that the buried Trp 310 residue is specifically involved in the interaction with acrylamide. This is possible without doing any site-directed mutagenesis of GAPD which contains Trp residues at 84, 193 and 310. Tyrosine 320 is also specifically quenched. The results have been corroborated using the molecular docking studies. Molecular Dynamics simulation supports our contention of the involvement of Trp 310 and also shows that the other nearest residues of acrylamide are Val175 and Val232.