Nanosecond time-dependent Stokes shift at the tunnel mouth of haloalkane dehalogenases.

The tunnel mouths are evolutionally the most variable regions in the structures of haloalkane dehalogenases originating from different bacterial species, suggesting their importance for adaptation of enzymes to various substrates. We decided to monitor the dynamics of this particular region by means of time-resolved fluorescence spectroscopy and molecular dynamic simulations. To label the enzyme specifically, we adapted a novel procedure that utilizes a coumarin dye containing a halide-hydrocarbon linker, which serves as a substrate for enzymatic reaction. The procedure leads to a coumarin dye covalently attached and specifically located in the tunnel mouth of the enzyme. In this manner, we stained two haloalkane dehalogenase mutants, DbjA-H280F and DhaA-H272F. The measurements of time-resolved fluorescence anisotropy, acrylamide quenching, and time-resolved emission spectra reveal differences in the polarity, accessibility and mobility of the dye and its microenvironment for both of the mutants. The obtained experimental data are consistent with the results obtained by molecular dynamics calculations and correlate with the anatomy of the tunnel mouths, which were proposed to have a strong impact on the catalytic activity and specificity of the examined mutants. Interestingly, the kinetics of the recorded time-dependent Stokes shift is unusual slow; it occurs on the nanosecond time-scale, suggesting that the protein dynamics is extremely slowed down at the region involved in the exchange of ligands between the active-site cavity and bulk solvent.

[1]  S. Sano,et al.  JAXA-GCF project – the past, present and future , 2007 .

[2]  M. Hargrove,et al.  Solvation dynamics in protein environments: comparison of fluorescence upconversion measurements of coumarin 153 in monomeric hemeproteins with molecular dynamics simulations. , 2007, The Journal of chemical physics.

[3]  B. Cohen,et al.  Measurement of solvation responses at multiple sites in a globular protein. , 2007, The journal of physical chemistry. B.

[4]  J. Bujnicki,et al.  Phylogenetic analysis of haloalkane dehalogenases , 2007, Proteins.

[5]  J. Koča,et al.  CAVER: a new tool to explore routes from protein clefts, pockets and cavities , 2006, BMC Bioinformatics.

[6]  K. Bhattacharyya,et al.  Femtosecond study of partially folded states of cytochrome C by solvation dynamics. , 2006, The journal of physical chemistry. B.

[7]  P. Jurkiewicz,et al.  Solvent Relaxation in Phospholipid Bilayers: Principles and Recent Applications , 2005, Journal of Fluorescence.

[8]  Lennart Nilsson,et al.  Molecular origin of time-dependent fluorescence shifts in proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Siddhartha Roy,et al.  Slow solvation dynamics at the active site of an enzyme: implications for catalysis. , 2005, Biochemistry.

[10]  Alexander D. MacKerell,et al.  Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations , 2004, J. Comput. Chem..

[11]  Dick B Janssen,et al.  Evolving haloalkane dehalogenases. , 2004, Current opinion in chemical biology.

[12]  Samir Kumar Pal,et al.  Dynamics of water in biological recognition. , 2004, Chemical reviews.

[13]  G. Otting,et al.  Dynamics of protein and peptide hydration. , 2004, Journal of the American Chemical Society.

[14]  Yuji Nagata,et al.  Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of Its Entrance Tunnel* , 2003, Journal of Biological Chemistry.

[15]  Siddhartha Roy,et al.  Solvation dynamics in the molten globule state of a protein , 2003 .

[16]  K. Bhattacharyya,et al.  Solvation dynamics in a protein-surfactant complex , 2003 .

[17]  Manoj Kumbhakar,et al.  Photophysical properties of coumarin-120: Unusual behavior in nonpolar solvents , 2003 .

[18]  T. C. Bruice,et al.  Comparison of formation of reactive conformers for the SN2 displacements by CH3CO2- in water and by Asp124-CO2- in a haloalkane dehalogenase. , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  K. Bhattacharyya.  Solvation dynamics and proton transfer in supramolecular assemblies. , 2003, Accounts of chemical research.

[20]  P. Kapusta,et al.  On What Time Scale Does Solvent Relaxation in Phospholipid Bilayers Happen , 2002 .

[21]  J. Newman,et al.  Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26. , 2000, Biochemistry.

[22]  Robin M. Hochstrasser,et al.  Ultrafast Dielectric Response of Proteins from Dynamics Stokes Shifting of Coumarin in Calmodulin , 2000 .

[23]  S. Dellerue,et al.  Relaxational dynamics of water molecules at protein surface , 2000 .

[24]  B. Bagchi,et al.  Dielectric relaxation and solvation dynamics of water in complex chemical and biological systems. , 2000, Chemical reviews.

[25]  M. Nardini,et al.  α/β Hydrolase fold enzymes : the family keeps growing , 1999 .

[26]  J. Newman,et al.  Haloalkane dehalogenases: structure of a Rhodococcus enzyme. , 1999, Biochemistry.

[27]  J. Damborský,et al.  Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26. , 1999, Protein expression and purification.

[28]  G. Fleming,et al.  Solvation Dynamics in Protein Environments Studied by Photon Echo Spectroscopy , 1999 .

[29]  M F Sanner,et al.  Python: a programming language for software integration and development. , 1999, Journal of molecular graphics & modelling.

[30]  David S. Goodsell,et al.  Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function , 1998, J. Comput. Chem..

[31]  F. W. Schneider,et al.  Binding of prothrombin and its fragment 1 to phospholipid membranes studied by the solvent relaxation technique. , 1998, Biochimica et biophysica acta.

[32]  M. Field,et al.  A Generalized Hybrid Orbital (GHO) Method for the Treatment of Boundary Atoms in Combined QM/MM Calculations , 1998 .

[33]  W. F. Beck,et al.  Protein-Matrix Solvation Dynamics in the α Subunit of C-Phycocyanin , 1996 .

[34]  J. Schanstra,et al.  Kinetics of halide release of haloalkane dehalogenase: evidence for a slow conformational change. , 1996, Biochemistry.

[35]  M. Maroncelli,et al.  Subpicosecond Measurements of Polar Solvation Dynamics: Coumarin 153 Revisited , 1995 .

[36]  F. Bright,et al.  Dynamics of acrylodan-labeled bovine and human serum albumin sequestered within aerosol-OT reverse micelles. , 1995, Analytical chemistry.

[37]  K. H. Kalk,et al.  Crystallographic analysis of the catalytic mechanism of haloalkane dehalogenase , 1994, Nature.

[38]  Graham R. Fleming,et al.  Femtosecond solvation dynamics of water , 1994, Nature.

[39]  M. Maroncelli,et al.  Estimating the time-zero spectrum in time-resolved emmsion measurements of solvation dynamics , 1994 .

[40]  Joel L. Sussman,et al.  The α/β hydrolase fold , 1992 .

[41]  James J. P. Stewart,et al.  MOPAC: A semiempirical molecular orbital program , 1990, J. Comput. Aided Mol. Des..

[42]  G Vriend,et al.  WHAT IF: a molecular modeling and drug design program. , 1990, Journal of molecular graphics.

[43]  M Karplus,et al.  Active site dynamics of ribonuclease. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[44]  M. Karplus,et al.  Active site dynamics in protein molecules: A stochastic boundary molecular‐dynamics approach , 1985, Biopolymers.

[45]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[46]  M. Karplus,et al.  CHARMM: A program for macromolecular energy, minimization, and dynamics calculations , 1983 .

[47]  Roger J.-B. Wets,et al.  Minimization by Random Search Techniques , 1981, Math. Oper. Res..

[48]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[49]  K. Wood,et al.  The HaloTag: a novel technology for cell imaging and protein analysis. , 2007, Methods in molecular biology.

[50]  A. Kulakova,et al.  The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064. , 1997, Microbiology.

[51]  J. Kingma,et al.  Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a Rhodococcus haloalkane dehalogenase , 2022 .