Using in situ tryptophan analogues to probe water micro-solvation in proteins via water catalyzed proton transfer reaction Water molecules inside protein, dubbed as bio-water, play a very important role in bioactivity, such as enzymatic reaction and molecular recognition and hence the signal transduction. Scientists have made tremendous efforts to gain understanding of the water molecules in proteins via indirect measurements such as molecular dynamic simulation and/or probing the polarity of the local environment. However, sensing bio-waters with a more direct method and hence their possible functionality in protein remains elusive. Recently, a team led by Prof. Chou in NTU Chemistry Department utilized a novel tryptophan analogue, 2,7-diazaindole tryptophan ((2,7-aza)Trp), to successfully probe the presence of water in proteins. This new tryptophan analog shows very unique properties. In neutral water, (2,7-aza)Trp exists in two proton-transfer isomers: The N(2)-H isomer exhibits a 380 nm emission band, and the N(1)-H isomer undergoes water catalyzed excited-state proton transfer (ESPT), giving an N(1)-H 340 nm emission band and a prominent green N(7)-H isomer 500 nm emission. These characteristic multiple emissions offer the unprecedented opportunity in sensing the water micro-solvation of proteins. They then studied a structurally undetermined protein “human thromboxane A2 synthase (TXAS)” by site-specifically replacing five Trp residues with this water sensitive bio-probe. The missing N(2)-H 380 nm emission indicates that water inside TXAS is not the “bulk-water” but is more like a “water-cluster” such that the equilibrium is prevailingly shifted to the N(1)-H isomer. After denaturing the protein, the N(2)-H 380 nm reappears, gives the future perspective that this probe can be used to study the structural dynamics of protein folding. More importantly, they observed only N(1)-H 350 emission but no green (500 nm) N(7)-H emission in (2,7-aza)Trp65 at the W65 site of TXAS. The result implies lack of ESPT and hence W65 site is water scant in its proximity. Conversely, as evidenced by the prominent green (500 nm) emission and thereby ESPT, the other four tryptophan sites are in water-contact environments. This team thus demonstrates a groundbreaking work in that water environment can be probed in protein without disrupting its native structure.1 The impact of this discovery is far-reaching. Relevant application has been in probing how water molecules affect the folding, structures and activities of proteins.2,3 References: 1. “Probing Water Micro-solvation in Proteins by Water Catalyzed Proton Transfer Tautomerism” Jiun-Yi Shen, Wei-Chih Chao, Chun Liu, Hsiao-An Pan, Hsiao-Ching Yang*, Chi-Lin Chen, Yi-Kang Lan, Li-Ju Lin, Jinn-Shyan Wang, Jyh-Feng Lu, Steven Chun-Wei Chou, Kuo-Chun Tang, Pi-Tai Chou, Nature Commun, 2013, 4, 2611. 2. “Probing Water Environment of Trp59 in Ribonuclease T1; Insight of the Structure-Water Network Relationship” Wei-Chih Chao, Jiun-Yi Shen, Jyh-Feng Lu, Jinn-Shyan Wang, Hsiao-Ching Yang, Kevin Wee, Li-Ju Lin, Yi-Ching Kuo, Cheng-Han Yang, Shih-Hui Weng, Huai-Ching Huang, You-Hua Chen, Pi-Tai Chou, J. Phys. Chem. B. 2015, 119 (6), pp 2157–2167. 3. “The In Situ Tryptophan Analogue Probes the Conformational Dynamics in Asparaginase Isozymes” Wei-Chih Chao, Jiun-Yi Shen, Cheng-Han Yang, Yi-Kang Lan, Jui-Hung Yuan, Li-Ju Lin, Hsiao-Ching Yang, Jyh-Feng Lu, Jinn-Shyan Wang, Kevin Wee, You-Hua Chen, Pi-Tai Chou, Biophysical. J. 2016, in press. About Figure Caption: The water-scant site of (2,7-aza)Trp (W65) in human thromboxane A2 synthase gives only normal 340 nm emission, whereas the water-rich site of (2,7-aza)Trp exhibits both normal 340 nm emission and green 500 nm emission. Accordingly, water distribution in protein can be mapped out via the ratiometric emission.