In this study, we used the tryptophan analogue, (2,7-aza)Trp, which exhibits water catalyzed proton transfer isomerization among N(1)-H, N(7)-H, and N(2)-H isomers, to probe the water environment of tryptophan-59 (Trp59) near the connecting loop region of ribonuclease Tl (RNase T1) by replacing the tryptophan with (2,7-aza)Trp. The resulting (2,7-aza)Trp59 triple emission bands and their associated relaxation dynamics, together with relevant data of 7-azatryptophan and molecular dynamics (MD) simulation, lead us to propose two Trp59 containing conformers in RNase T1, namely, the loop-close and loop-open forms. Water is rich in the loop-open form around the proximity of (2,7-aza)Trp59, which catalyzes (2,7-aza)Trp59 proton transfer in the excited state, giving both N(1)-H and N(7)-H isomer emissions. The existence of N(2)-H isomer in the loop-open form, supported by the MD simulation, is mainly due to the specific hydrogen bonding between N(2)-H proton and water molecule that bridges N(2)-H and the amide oxygen of Pro60, forming a strong network. The loop-close form is relatively tight in space, which squeezes water molecules out of the interface of a-helix and beta 2 strand, joined by the connecting loop region; accordingly, the water-scant environment leads to the sole existence of the N(1)-H isomer emission. MD simulation also points out that the Trp-water pairs appear to preferentially participate in a hydrogen bond network incorporating polar amino acid moieties on the protein surface and bulk waters, providing the structural dynamic features of the connecting loop region in RNase T1.