The In Situ Tryptophan Analogue Probes the Conformational Dynamics in Asparaginase Isozymes.
Dynamic water solvation is crucial to protein conformational reorganization and hence to protein structure and functionality. We report here the characterization of water dynamics on the L-asparaginase structural homology isozymes L-asparaginases I (AnsA) and II (AnsB), which are shown via fluorescence spectroscopy and dynamics in combination with molecular dynamics simulation to have distinct catalytic activity. By use of the tryptophan (Trp) analog probe 2,7-diaza-tryptophan ((2,7-aza)Trp), which exhibits unique water-catalyzed proton-transfer properties, AnsA and AnsB are shown to have drastically different local water environments surrounding the single Trp. In AnsA, (2,7-aza)Trp exhibits prominent green N(7)-H emission resulting from water-catalyzed excited-state proton transfer. In stark contrast, the N(7)-H emission is virtually absent in AnsB, which supports a water-accessible and a water-scant environment in the proximity of Trp for AnsA and AnsB, respectively. In addition, careful analysis of the emission spectra and corresponding relaxation dynamics, together with the results of molecular dynamics simulations, led us to propose two structural states associated with the rearrangement of the hydrogen-bond network in the vicinity of Trp for the two Ans. The water molecules revealed in the proximity of the Trp residue have semiquantitative correlation with the observed emission spectral variations of (2,7-aza)Trp between AnsA and AnsB. Titration of aspartate, a competitive inhibitor of Ans, revealed an increase in N(7)-H emission intensity in AnsA but no obvious spectral changes in AnsB. The changes in the emission profiles reflect the modulation of structural states by locally confined environment and trapped-water collective motions.
Department of Chemistry, Fu Jen Catholic University, New Taipei City, Taiwan.
Wei-Chih
WC
Chao
Department of Chemistry and Center for Emerging Material and Advanced Devices, National Taiwan University, Taipei, Taiwan.
Jiun-Yi
JY
Shen
Department of Chemistry, Fu Jen Catholic University, New Taipei City, Taiwan.
Cheng-Han
CH
Yang
Department of Chemistry, Fu Jen Catholic University, New Taipei City, Taiwan.
Yi-Kang
YK
Lan
Department of Chemistry and Center for Emerging Material and Advanced Devices, National Taiwan University, Taipei, Taiwan.
Jui-Hung
JH
Yuan
School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan.
Li-Ju
LJ
Lin
Department of Chemistry, Fu Jen Catholic University, New Taipei City, Taiwan. Electronic address: hcyang_chem@mail.fju.edu.tw.
Hsiao-Ching
HC
Yang
School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan.
Jyh-Feng
JF
Lu
School of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan.
Jinn-Shyan
JS
Wang
Department of Chemistry and Center for Emerging Material and Advanced Devices, National Taiwan University, Taipei, Taiwan.
Kevin
K
Wee
Department of Chemistry and Center for Emerging Material and Advanced Devices, National Taiwan University, Taipei, Taiwan.
You-Hua
YH
Chen
Department of Chemistry and Center for Emerging Material and Advanced Devices, National Taiwan University, Taipei, Taiwan. Electronic address: chop@ntu.edu.tw.
Pi-Tai
PT
Chou
D001215:Asparaginase / Q000737:chemistry* / Q000378:metabolism; D055162:Biocatalysis; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D007527:Isoenzymes; D014867:Water; D014364:Tryptophan; D001215:Asparaginase