document: electrostatic 
   interactions have been shown to play a vital role 
   in enzyme catalysis . 
   these interactions assist in the binding of ligands 
   and facilitate the enzyme - catalyzed chemical reactions by keeping 
   the reacting species positioned properly as well as stabilizing the 
   transition state in some cases . 
   moreover , these electrostatic interactions are expected to be significantly 
   modulated by the conformational changes that occur over the catalytic 
   cycle . in particular , the electrostatic environment in the 
   active site is likely to change as the ligands bind , the chemical 
   reaction occurs , and the ligands are released . quantifying the electrostatic 
   contributions to the individual catalytic steps , as well as 
   the impact 
   of conformational changes on the electrostatic interactions , is a 
   challenging yet important goal in the field of enzyme catalysis . to gain a better understanding of the catalytic role of electrostatics 
   and conformational motions 
   , we probed the changes in the active site 
   electrostatic environment resulting from structural rearrangements 
   that occur along the catalytic cycle of escherichia 
   coli dihydrofolate reductase ( ecdhfr ) . 
   this enzyme catalyzes the nadph - dependent conversion of 7,8-dihydrofolate 
   ( dhf ) to 5,6,7,8-tetrahydrofolate ( thf ) . 
   x - ray crystallographic , nuclear 
   magnetic resonance ( nmr ) , theoretical , and photophysical studies have 
   shown that ecdhfr exhibits significant structural 
   rearrangements along the catalytic cycle . 
   thus , ecdhfr is a highly suitable 
   system for studying how conformational changes can affect the active 
   site electrostatic environment , which can then modulate enzyme catalysis . 
   recently , thiocyanate and other vibrational probes have been incorporated 
   into a number of biological systems to investigate the local electrostatic 
   environments . 
   boxer and co - workers have explored the use of nitrile ( cn ) probes 
   to detect differences in the electric field projected along the cn 
   axis in proteins upon ligand binding , protein folding , and protein  protein 
   association . using this method 
   , they showed that the electrostatic 
   environment in the active site of -3-ketosteroid 
   isomerase ( ksi ) exhibits limited rearrangement upon photoexcitation 
   of a ligand , where this excitation was proposed to mimic the movement 
   of charge during the catalyzed chemical transformation . 
   the authors concluded that the ksi active site 
   is electrostatically preorganized for catalysis in its tertiary protein 
   structure and that major rearrangement is not necessary for catalysis . 
   this observation is not unexpected due to the relatively rigid structure 
   of ksi that limits substantial conformational mobility , 
   thereby limiting electrostatic rearrangement . 
   however , calculations 
   have shown that further active site reorganization , manifesting as 
   small conformational changes that position the reacting species for 
   chemistry , is still required in ksi to enable the catalyzed proton 
   transfer reactions . 
   in contrast to ksi , 
   which exhibits limited protein motion along the catalytic cycle , ecdhfr is known to undergo significant conformational changes 
   along the catalytic cycle , suggesting that larger changes in electrostatics 
   could play a more significant role in all steps of the catalytic cycle . to examine the electrostatic interactions within the active site 
   of ecdhfr , we incorporated thiocyanate probes at 
   site - specific locations . using vibrational and c nmr 
   spectroscopic techniques , as well as computer simulations , we analyzed 
   the electrostatics and degree of hydration of the microenvironments 
   surrounding the probes . 
   the electrostatic interactions arising from 
   the active site microenvironments surrounding the small , catalytically 
   nonintrusive probes led to different observed cn vibrational stretching 
   frequencies and c nmr chemical shifts along the catalytic 
   cycle . 
   experimental data clearly show changes in the active site electrostatic 
   environment due to structural rearrangements . 
   further insights were 
   obtained through mixed quantum mechanical and molecular mechanical 
   ( qm / mm ) analyses of classical molecular dynamics ( md ) simulations . 
   these calculations were able to reproduce the experimentally measured 
   vibrational frequency shifts reported by the probes across the enzyme - catalyzed 
   hydride transfer step of the catalytic cycle . 
   moreover , 
   the electrostatic contributions from the active site microenvironments 
   surrounding the probe were decomposed into the major components ( individual 
   residues and ligands ) . 
   in addition , the experimentally validated theoretical 
   model allowed us to examine the electric field along the hydride transfer 
   pathway between the substrate and cofactor , a property that is catalytically 
   important yet unattainable experimentally . 
   this integrated approach 
   provides valuable insights into the interplay between electrostatic 
   and conformational changes along the catalytic cycle of ecdhfr . 
   7,8-dihydrofolate 
   ( dhf ) and [ ( 4r)-h]nadph 
   ( nadpd ) were prepared 
   according to previously described procedures . 
   -nicotinamide 
   adenine dinucleotide phosphate reduced tetra(cyclohexylammonium ) salt 
   ( nadph ) , -nicotinamide adenine dinucleotide phosphate hydrate 
   ( nadp ) , potassium cyanide - c ( 99 atom % c ) , methotrexate , methotrexate - agarose , dithiothreitol ( dtt ) , 
   folic acid ( 97% ; fol ) , sodium phosphate monobasic monohydrate 
   ( 98102% ) , and 5,5-dithiobis(2-nitrobenzoic acid ) ( ellman s 
   reagent ) were purchased from sigma - aldrich and used without further 
   purification . 
   sodium phosphate dibasic 
   heptahydrate and 2-nitro-5-thiocyanatobenzoic acid ( 97% ; ntcb ) were 
   purchased from emd and tci america , respectively . 
   ph values were measured 
   using an accumet model 13 - 620 - 300 standard combination electrode calibrated 
   with vwr certified standard aqueous buffers ( ph = 4 , 7 , and 10 ) . 
   all 
   of the experiments , including kinetics and spectroscopic measurements , 
   were done in ph 7.0 50 mm sodium phosphate buffer unless otherwise 
   specified . 
   first , the two native cysteines ( c85 and 
   c152 ) in wild - type ( wt ) ecdhfr were mutated to ala 
   and ser , respectively , to generate 
   cys ecdhfr using the stratagene quikchange 
   site - directed mutagenesis kit and the wt ecdhfr template 
   as described elsewhere . 
   primer sequences 
   were 5-c gaa gcc atc gcg gcg gct ggt 
   gac gta cca g-3 ( c85a ) and 5-c tcg cat agc tat tca ttc gaa atc ctc g-3 ( c152s ) . 
   the choice of 
   amino acid substitution was based on a previous report showing that 
   the c85a / c152s substitution on ecdhfr induced minimal 
   perturbation to the enzymatic activity . 
   selective incorporation of cysteine was achieved through subsequent 
   mutations using the following primers : 5-ca atc ggt agg cct tgc ccc ggc cgc aaa aat att atc-3 ( l54c ) and 
   5-g att atg ggg cgc cat tgc tgg gaa 
   tca atc g-3 ( t46c ) . 
   plasmid construction , protein expression , 
   and purification of mutant dhfrs were performed according to published 
   protocols . 
   labeling of the single - cysteine ecdhfr constructs 
   ( cys l54c and cys t46c , denoted as l54c - cn and t46c - cn , 
   respectively ) was achieved using a modified version of a published 
   procedure . inside an amicon 
   ultra-4 10k 
   nominal molecular weight limit ( nwml ) centrifugal filter tube , approximately 
   400 m of each ecdhfr construct was mixed with 
   four times excess of ntcb in 4 ml of 0.1 m sodium phosphate buffer 
   at ph 7.4 . 
   the mixture was allowed to react at room temperature for 
   10 min , and reaction progress was monitored by the appearance of the 
   phenolic product using a uv  vis spectrophotometer . 
   the protein 
   mixture was then concentrated by spinning at 5k rpm in a centrifuge 
   at 4 c . 
   the protein sample was then washed twice with 12 mm 
   kcn in ph 7.0 50 mm sodium phosphate buffer , with a concentration 
   step after each wash . 
   this was followed by washing the protein sample 
   with 50 mm ph 7.0 sodium phosphate buffer two more times before concentrating 
   the protein solution down to 0.8 ml , which was then applied 
   onto a size exclusion column ( econo - pac 10dg ; bio - rad ) that was pre - equilibrated 
   with ph 7.0 50 mm sodium phosphate buffer . 
   the isolated labeled proteins 
   were concentrated in an amicon ultra-4 10k nmwl centrifugal filter 
   tube . 
   the intact masses of scn labeled and unmodified samples were 
   confirmed by esi - ms , which yield deconvoluted m / z values of 17965 ( l54c)/17978 ( t46c ) and 17940 ( l54c)/17953 
   ( t46c ) da , respectively . 
   the theoretical molecular weights for the 
   unmodified samples are 17941 ( l54c)/17953 ( t46c ) da . 
   similar 
   preparation procedures were used for the scn labeled 
   samples . a mixture containing 10 mm ellman 
   s reagent and 0.5 
   m kcn in ph 7.0 0.1 m sodium phosphate buffer was prepared 
   at room temperature for 15 min . 
   this mixture was then allowed to react 
   with a sample of the unlabeled protein in an amicon ultra-4 10k nmwl 
   for 10 min at room temperature . 
   the final concentrations of a typical 
   reaction mixture were 500 m protein , 5 mm ellman s reagent , 
   and 0.25 m kcn . 
   the subsequent purification and isolation 
   steps were similar to those for scn labeling , except that kcn was used for washes rather than kcn . 
   successful labeling was confirmed 
   spectroscopically by the release of one equivalent of the phenolic 
   product as well as c nmr comparison with the unlabeled 
   proteins . the pre - steady - state 
   hydride transfer reaction was monitored on an applied photophysics 
   stopped - flow spectrophotometer thermostated at 25 c . 
   all of 
   the enzymatic reactions were performed in 50 mm sodium phosphate buffer 
   ph 7.0 . 
   one of the syringes in the stopped - flow analyzer was loaded 
   with 20 m enzyme , 250 m nadph , and 2 mm dtt . 
   after combining dhfr 
   and nadph as described above , the mixtures were prepared on ice for 
   5 min prior to the onset of the chemical reaction . upon mixing , 
   the 
   final concentrations of the individual species in the reaction chamber 
   were halved . 
   the conversion of dhf to thf was followed by the loss 
   of fluorescence resonance energy transfer from the enzyme to nadph . 
   the reaction mixture was excited at 290 nm , and the emission was measured 
   using a 400 nm cutoff output filter . 
   the 
   measure of absorbance vs time trace ( of burst phase ) was fit to standard 
   single exponential decay to obtain the hydride transfer rate ( khyd ) . 
   the kinetic isotope effect ( kie ) for the 
   hydride transfer rate was also determined under the same experimental 
   conditions , with the exception that nadpd was used instead of nadph 
   in comparative experiments . 
   steady - state enzyme turnover kinetics 
   ( kcat ) were studied following similar 
   experimental conditions , with the exception that the reaction progress 
   was monitored at 340 nm . under each condition , at least 5 independent 
   kinetic runs were conducted , and the average rate constants were used 
   for analysis . 
   ftir spectra were 
   obtained at room temperature using a mattson research series ftir 
   spectrometer ( madison instruments , inc . ) 
   equipped with a liquid - nitrogen 
   cooled hgcdte ( mct ) detector ( infrared associates , inc . ) . cyanylated 
   dhfr samples ( [ protein ] = 1.72.5 mm ) were mixed with appropriate 
   ligands ( 1:10 mol equiv ) in aqueous buffer and placed in a liquid 
   cell with two caf2 windows separated by a 56 m teflon 
   spacer . 
   all nitrile ( cn ) stretch absorbance spectra for cyanylated 
   dhfr samples consisted of 2000 scans and the data were collected with 
   1.0 cm resolution . 
   the cn stretch absorbance spectra 
   for cyanylated dhfr samples were obtained by subtracting a spectrum 
   of the aqueous buffer solution from the protein spectrum . 
   the baseline 
   for each spectrum was corrected by fitting a fifth - order polynomial 
   fit to the data , as done previously by fafarman et al . and mcmahon et al . 
   the fifth - order polynomial local fit functions were obtained by 
   defining roots at least 15 cm from the peak maximum , 
   meaning the experimental data and fits were forced to be equal at 
   points more than 15 cm from the central frequency . 
   all published spectra are the averages of two independently prepared 
   protein samples after the baseline has been corrected and the peak 
   height has been normalized to one . 
   the peak positions obtained from 
   independently prepared protein samples typically differed by < 0.5 
   cm , which is substantially less than the variation 
   among peak positions due to the protein binding or releasing the ligands . 
   detailed experimental setup and data analysis are provided in the supporting information . 
   c nmr spectra were 
   collected on a bruker av - iii-600 ( 150.9 
   mhz c frequency ) at 25 c . 
   samples were prepared 
   with 1.6 mm of the scn labeled protein , 8 mm ligands , 
   and 50 mm potassium phosphate ( ph 7.0 ) in water . 
   a sealed capillary 
   tube containing 1 m acetone in d2o was inserted into the 
   nmr tube that holds the protein sample . 
   the 1d c 
   nmr spectral data obtained for each sample were compared with its 
   corresponding c dept 45 ( distortionless enhancement 
   by polarization transfer ) data to confirm the identity of the assigned 
   peaks . under the same experimental conditions , we were unable to detect 
   the c nmr signals corresponding to the scn probes for 
   samples containing either the nonisotopically enriched scn label or 
   the unlabeled proteins . 
   crystals 
   of e. coli t46c - cn and l54c - cn dhfr 
   variants were obtained by the hanging - drop vapor diffusion method 
   using conditions similar to those previously described . 
   detailed methods , data collection , and refinement 
   statistics are provided in the supporting information . 
   we calculated 
   the ir spectra for the cn stretching mode of the incorporated thiocyanate 
   probes using a two - step method . 
   first we propagated classical md trajectories 
   for the entire solvated enzyme with bound ligands . 
   the configurations 
   sampled from the md trajectory were then analyzed using a series of 
   qm / mm single point energy calculations to determine the cn stretching 
   potential energy curve . 
   the qm region was treated with a semiempirical 
   method that was reparameterized for the calculation of nitrile vibrational 
   frequencies , and the mm region was represented as a collection of 
   atomic point charges . 
   the instantaneous vibrational wave functions 
   and associated frequencies from the energy level splittings were obtained 
   from the cn stretching potential energy curve using a grid - based numerical 
   approach . 
   the simulated ir spectra were calculated using the fluctuating 
   frequency approximation , which includes the dynamical contributions 
   of the protein motion to the simulated lineshapes . 
   a full description of the methodology can be found in our 
   previous application to ksi , with some 
   minor adjustments that will be discussed below . 
   we propagated classical 
   md trajectories for the cysteine - free t46c - cn and l54c - cn ecdhfr mutants using the gromacs program . 
   the first state has folate ( fol ) bound 
   in the substrate pocket and the nicotinamide adenine dinucleotide 
   phosphate cofactor bound in the oxidized form ( nadp ) . 
   in this state , 
   the met20 loop is in the closed conformation , which 
   is thought to facilitate the hydride transfer reaction by positioning 
   the ligand and cofactor while excluding solvent molecules . 
   this state has been shown to structurally mimic 
   the reactive michaelis complex ( e : dhf : nadph ) prior to hydride transfer . although we can simulate the reactive michaelis 
   complex without hydride transfer occurring , we simulated the e : fol : nadp ternary complex to compare directly with the experimentally 
   measured ir spectra . 
   the second state that we simulated has the product 
   thf and nadp bound . in this state , 
   the met20 loop is in 
   the occluded conformation , and the nicotinamide ring of nadp is excluded from the active site . 
   these two states are used to model 
   the closed to occluded transition that occurs across the hydride transfer 
   step of the ecdhfr catalytic cycle . 
   the initial 
   structures for the md simulations were obtained from crystal structures 
   solved with the nitrile probes included for both the l54c - cn and t46c - cn 
   variants ( pdb accession codes 4p68 and 4p66 , respectively ) . 
   the crystal structures 
   have methotrexate bound in the substrate binding pocket and nadph 
   bound in the active site . in both of these structures , 
   this state is thought to mimic the 
   transition state for the hydride transfer reaction . 
   for the simulations in the closed conformation ( nadp / fol ) , the folate molecule was incorporated by aligning the 
   folate substrate with the corresponding heavy atoms of the methotrexate 
   ligand from the crystal structure . for the simulations in the occluded 
   conformation ( nadp / thf ) 
   , we used the initial coordinates 
   of a wt crystal structure of ecdhfr complexed with 
   nadph and 5,10-dideazatetrahydrofolate ( ddthf ) ( pdb 1rx6 ) . 
   the thiocyanate probes were incorporated into the occluded 
   structure by aligning the backbone atoms of the five previous and 
   five subsequent residues to the cyanylated cysteine residue in the 
   new crystal structure that contains the probe to the corresponding 
   backbone atoms of the 1rx6 structure . 
   we then replaced the specific residue with 
   the aligned cyanalated cysteine and optimized the probe residue geometry 
   and position using a steepest - descent algorithm in gromacs , while 
   the ligands , the crystallographic water molecules , and the rest of 
   the enzyme were held fixed . 
   the substrate ligand ( ddthf ) was replaced 
   by thf by aligning the corresponding heavy atoms in ddthf and thf , 
   and the oxidized cofactor was modeled by aligning the heavy atoms 
   of nadp with the nadph from the crystal structure . 
   the 
   modeled ligands were also optimized using the steepest - descent algorithm 
   with the enzyme and the crystallographic water molecules held fixed . 
   for all of the md simulations , we used the amber99sb force field 
   to compute the potential energy and forces for the protein . 
   the bonded and nonbonded parameters for the nadp cofactor were consistent with those from our previous 
   work . 
   the fol and thf charges were calculated 
   using the restrained electrostatic potential ( resp ) method , and the bonded and van der waals parameters 
   were obtained from the generalized amber force field ( gaff ) . for methyl thiocyanate and the cyanylated cysteine 
   residue , the bonded parameters were obtained from cho et al . , and the nonbonded parameters were obtained from 
   lindquist and corcelli , as used in our 
   previously published work on ksi . 
   each of the enzymatic systems was solvated with tip3p water in a truncated octahedral box , where the sides 
   of the box were at least 10.0  from every atom in the enzymatic 
   complex , including the crystallographic waters . 
   after solvation , we 
   added 12 sodium atoms using the gromacs genion utility 
   to neutralize the system before simulation . 
   all bonds involving a 
   hydrogen atom were constrained to their equilibrium bond lengths using 
   the lincs algorithm as implemented in gromacs . 
   nonbonded van der waals interactions were calculated with 
   a cutoff of 1.0 nm , and electrostatic interactions were calculated 
   using the particle - mesh ewald method . after the initial preparation of each system , we followed 
   a well - defined 
   equilibration procedure . 
   the solvent configurations were minimized 
   using the steepest - descent algorithm in gromacs with the enzyme and 
   ligands held fixed to mitigate any possible bad contacts between the 
   solvent and solute molecules . 
   the solvent molecules and counterions 
   were then equilibrated for 50 ps at 300 k in the canonical ( constant 
   nvt ) ensemble with the protein and ligands still fixed . 
   after solvent 
   equilibration , the entire system was then minimized using the steepest - descent 
   algorithm . 
   subsequently , the system was equilibrated using a simulated 
   annealing procedure in which the initial velocities were generated 
   according to a boltzmann distribution at 50 k. the system was equilibrated 
   in the isothermal  isobaric ensemble ( constant npt ) at 50 k 
   for 50 ps , and then the temperature was increased up to 300 k in increments 
   of 50 k with 50 ps of equilibration at each temperature . 
   after this 
   simulated annealing procedure , an additional 1 ns of equilibration 
   in the constant npt ensemble was performed , and finally 1 ns of equilibration 
   in the constant nvt ensemble was performed . during the simulations , 
   the nos  hoover thermostat was used to maintain the temperature , and the parrinello 
   the production trajectories were propagated in 
   the canonical ensemble 
   at 300 k with a time step of 1 fs and a total simulation time of 1 
   ns for each independent trajectory . 
   configurations were saved at every 
   time step and were analyzed using the qm / mm methodology described 
   below . for each mutant and catalytic state , we propagated at least 
   two independent trajectories with different initial configurations . 
   the data for a single trajectory are presented in the main paper , 
   while data from additional trajectories are provided in the supporting information . 
   the results for independent 
   trajectories associated with the same system were found to be consistent 
   within the numerical errors of the methodology . 
   the methodology 
   used to calculate the vibrational frequency of the 
   nitrile group is similar to that used in our previous work . here 
   we highlight the differences between the 
   methods used in our previous work and the methods employed in the 
   present work . as in our previous work , we calculated the vibrational 
   frequency by generating the one - dimensional potential energy curve 
   along the cn stretching mode using a qm / mm method . the potential energy 
   is calculated along the cn vibrational coordinate by freezing all 
   of the atomic centers in the system and moving only the nitrogen atom 
   to stretch the cn distance from 1.0 to1.4  with a step size 
   of 0.05  . 
   the qm calculations were performed with the mm region 
   treated as atomic point charges that polarize the qm region . 
   these 
   atomic point charges were the same as those used in the md simulations 
   and were obtained from the amber99sb force field for the standard 
   amino acid residues , the tip3p force field for the water molecules , 
   and the resp calculations for the ligands . 
   the choice of the qm region 
   for each probe studied in this work will be discussed below . in our 
   previous work , 
   the one - dimensional potential 
   energy curve for the cn stretching mode was fit to a morse function , 
   and the energy levels were calculated analytically from the morse 
   parameters . in the present work , 
   the potential energy curve was fit 
   to a third - order b - spline potential , and 
   the vibrational wave functions and energy levels were calculated numerically 
   using the fourier grid hamiltonian ( fgh ) method . 
   an advantage of the fgh method is that it does not assume any functional 
   form of the potential energy curve and therefore avoids the bias associated 
   with fitting to the morse function , which depends on only three parameters . in principle 
   , any electronic structure method can be used to calculate 
   the vibrational stretching potential , but the level of theory must 
   be computationally tractable for the millions of configurations that 
   are required to obtain statistically converged ir spectra . as in our 
   previous work , we used a reparameterized version of the semiempirical 
   pm3 method . in this semiempirical method , 
   some of the computationally expensive integrals in hartree 
   these empirical parameters can be varied to reproduce 
   properties from either electronic structure theory calculations or 
   experimental measurements . because we altered our approach to use 
   the fgh numerical method to calculate the vibrational frequencies , 
   we reoptimized the pm3 parameters . 
   the reparameterization procedure 
   is similar to that from our previous studies , and a full description 
   is provided in the supporting information . 
   we also confirmed that the new set of parameters reproduces the 
   results for ksi from our previous work when the fgh numerical method 
   is used for calculating the vibrational frequencies . 
   table s6 provides the calculated frequencies for the two different 
   vibrational probes in ksi that were studied previously . to assist 
   in the analysis of the vibrational frequency calculations 
   , we calculated 
   the average electric field at the midpoint of the cn bond in the thiocyanate 
   group from our md simulations . 
   the vibrational stark effect between 
   two states is described by1here h is 
   planck s 
   constant , cn is the shift in vibrational 
   frequency between the two states , eenv is the difference in the electric field vector at 
   the nitrile bond as a result of the change in the environment , cn is the difference dipole moment 
   for the vibrational mode that lies along the cn bond axis , and  
   is the angle between these two vectors . the magnitude of cn is the stark tuning rate , which 
   is a measure of the susceptibility of the probe to changes in the 
   local electric field . 
   boxer and co - workers have measured the stark 
   tuning rate for thiocyanate probes in various environments , including 
   in proteins , to be 0.7 cm/(mv / cm ) . 
   we emphasize that this equation accounts only 
   for the classical electrostatic effects of the environment and does 
   not account for the quantum mechanical aspects of effects such as 
   specific hydrogen - bonding interactions and polarization . 
   while these 
   phenomena have significant classical components , they also have nonclassical 
   aspects that can be incorporated into the calculations by treating 
   interacting residues quantum mechanically , as will be discussed below . 
   recent work explores the role of a local field correction factor that scales the externally applied electric 
   field to obtain the local field experienced by the probe , which is 
   typically larger because of the polarization of the local environment 
   induced by the applied electric field . 
   this local field correction 
   factor has been estimated to be between 1.1 and 1.3 for protein environments , but recent investigations have suggested that it may be as large 
   as 1.8 . 
   we have not considered the role 
   of the local field correction factor in our analysis of the electric 
   fields in dhfr due to the lack of consensus about its value for a 
   protein environment . 
   moreover , applying the local field corrections 
   would result in changes that are within the error bars of our calculations . to calculate the electric field at the nitrile bond , 
   a virtual 
   particle was positioned at the midpoint of the cn axis during the 
   md trajectory . 
   this virtual particle had no electrostatic or van der 
   waals interactions with other particles , and the bond , angle , and 
   dihedral parameters were set to zero . 
   the configurations from the 
   md trajectory were saved , and subsequently the forces acting on all 
   particles were recalculated for these configurations when the charge 
   on the virtual particle was set to + 1 . 
   the electric field , evp , at the virtual particle location 
   was calculated as2where fvp is the force acting on the virtual particle and qvp is the charge of the virtual particle . 
   the total electric 
   field vector , as well as the component along the cn axis , was calculated 
   for each configuration along the md trajectory . 
   calculation of the 
   component of the electric field along the cn axis allows us to calculate 
   the change in the vibrational frequency due to classical electrostatics 
   using eq 1 . 
   the remaining frequency change can 
   be attributed to the quantum mechanical aspects of specific hydrogen - bonding 
   interactions and polarization effects . 
   these effects can be investigated 
   by using a classical polarizable force field or by treating the interacting residues 
   quantum mechanically . 
   note that the experimentally measured and theoretically 
   calculated frequency shifts can be compared directly , but the breakdown 
   into the classical electrostatics and quantum mechanical aspects is 
   available only from the theoretical calculations . 
   we also analyzed 
   the contributions of individual residues and ligands 
   to the electric field at the midpoint of the cn bond . because the 
   classical electric field is the sum of the individual contributions 
   from each atom , the electric field 
   analysis of the contributions 
   from specific residues provides insight into the impact of conformational 
   changes occurring during the closed to occluded transition associated 
   with hydride transfer . 
   we generated 
   the t46c - cn and l54c - cn mutants by site - specifically incorporating 
   a thiocyanate probe at each position ( figure 1 ) . 
   the t46c - cn probe is situated in close proximity to the junction 
   between the two reacting ligands , dhf and nadph , allowing us to monitor 
   the microenvironment close to the reaction center . 
   the l54c - cn probe is located 9  from the 
   center of the hydride donor  acceptor ( d  a ) axis . 
   this 
   probe monitors the folate binding pocket , and its spectral character 
   is expected to respond to the formation of the ternary michaelis 
   menten 
   complex upon folate binding , as well as the release of thf in the 
   enzyme turnover step . 
   ( a ) superposition of the t46c - cn and l54c - cn ecdhfr mutants in the closed conformation with folate and nadp bound , where only the thiocyanate residue is shown for the 
   l54c - cn mutant . 
   ( b ) ecdhfr in the closed ( red ) and 
   the occluded ( blue ) conformations exhibited by the ( c ) five major 
   complexes in its catalytic cycle . 
   the enzyme activity of the two ecdhfr variants 
   was evaluated by their ability to catalyze the nadph - dependent reduction 
   of dhf and the enzyme turnover rates at ph 7.0 and 25 c . 
   the 
   catalytic efficiencies of the t46c - cn ( khyd = 120  10 s ; kcat = 6.5  0.5 s ) and l54c - cn ( khyd = 250  20 s ; kcat = 28  3 s ) variants were 
   comparable to that of wt ecdhfr ( khyd = 220 s ; kcat = 12 s ) . 
   we also found 
   the cys ecdhfr ( c85a / c152s ; khyd = 215  8 s ; kcat = 10.3  0.8 s ) variant to 
   exhibit activity similar to the wt enzyme . 
   furthermore , figure 
   s1 shows that the crystal structures of the ternary complexes 
   of the t46c - cn and l54c - cn mutants in the closed conformation overlap 
   well with the wt crystal structure . 
   these observations suggest that 
   the incorporation of the scn probes into the selected locations of 
   the active site does not significantly alter the structure or activity 
   of the enzyme . 
   it should be noted that we also incorporated 
   the scn probe at the 
   i50 position ( cys - i50c - cn ) . 
   however , such a modification decreased 
   the hydride transfer rate by 10 times at ph 7.0 and 25 c . 
   we felt that the degree of enzyme activity reduction in cys - i50c - cn 
   might result in a less accurate representation of the electrostatic 
   environment in the active site of the wt enzyme . 
   thus , further investigations 
   were not performed for the cys - i50c - cn construct . 
   it is expected 
   that even small modifications to the crowded active site can have 
   large effects on enzyme activity . 
   the ecdhfr 
   catalytic cycle contains the five major species ( figure 1c ) that reflect the significant conformational changes in 
   the active site met20 loop ( figure 1b ) . 
   menten 
   complex ( e : nadph : dhf ) to an occluded conformation in the initial ternary 
   product complex ( e : nadp : thf ) . 
   the enzyme remains in the 
   occluded conformation in the two subsequent product - containing species 
   ( e : thf and e : nadph : thf ) , until the met20 loop reverts back to the 
   closed conformation upon the rate - limiting release of thf to generate e : nadph ( figure 1c ) . 
   nmr relaxation dispersion experiments have found that the rates 
   of the met20 loop conformational change are temporally similar to 
   their corresponding enzymatic processes . 
   in other words , the hydride transfer and enzyme turnover rates are 
   similar to the values determined from the nmr experiments for the 
   conformational transitions that connect the relevant enzyme complexes . 
   while the major change in the met20 loop conformation appears to be 
   induced by the formation of the product thf 
   , theoretical simulations have shown that minor structural rearrangements 
   of the ligands and protein , not restricted to the met20 loop , are 
   necessary to progressively optimize the substrate orientations and 
   the active site environment along the reaction coordinate of the hydride 
   transfer reaction . 
   herein , we utilized the scn probes 
   to experimentally monitor the changes in the local microenvironments 
   in two locations in the active site over the five major catalytic 
   species . 
   the five complexes that constitute the catalytic cycle 
   of ecdhfr ( figure 2 ) were 
   generated using 1.52.5 mm of the enzyme and at least five 
   times excess of the ligands in ph 7.0 phosphate buffer . 
   these concentrations 
   should be more than sufficient to push the equilibria toward full 
   complexation , thus allowing us to obtain vibrational ftir spectra of the five 
   major complexes that constitute the ecdhfr catalytic 
   cycle , as well as for the apoenzyme and the e : nadph : fol complex . 
   note 
   that the e : nadph : dhf state is modeled by e : nadp : fol , as 
   discussed in the literature . 
   for each 
   enzyme complex , the ir spectrum was obtained in duplicate / triplicate 
   using separately prepared samples , and these spectra were found to 
   be very reproducible ( data in supporting information ) . after correcting the ir data for baseline and subsequently normalizing 
   the amplitude of the peak to 1.0 
   , we determined the maximum frequency 
   ( max ) from the first derivative of the corrected 
   ir peak . 
   the peak position is a measure of the transition energy between 
   the vibrational ground state and first excited state of the thiocyanate 
   probe . 
   the transition energy is sensitive to the electric field along 
   the cn bond because the vibrational potential energy function is perturbed 
   by the local electrostatic environment ( surrounding residues , ligands , 
   and solvent molecules ) which modulates the energy difference between 
   the vibrational quantum states . 
   color coding the frequency of maximum 
   absorption detected in each complex illustrates the changes in the 
   local electrostatic interactions experienced by the cn probe over 
   the catalytic cycle ( figure 2 ) . 
   ( b ) 
   nmr ( scn ) measurements along 
   the catalytic cycle of ecdhfr . 
   the color scales for 
   the ftir and nmr data have units of cm and ppm , 
   respectively . 
   the cofactor is orange , and the substrate / product is 
   purple in all complexes ( the coloring of the cofactor and substrate / product 
   does not correspond to the color scales that represent the nmr shift 
   or ir frequency ) . 
   enzyme complex labels in blue are in the occluded 
   conformation , and those in red are in the closed conformation . 
   the 
   measured values for ir frequency and nmr chemical shift are provided 
   in table s4 . 
   figure 2 indicates that the local 
   environment 
   of each cn probe changes significantly over several key steps in the 
   catalytic cycle : formation of the ternary michaelis 
   menten 
   model ground state complex , the hydride transfer step , and the rate - limiting 
   release of thf . 
   the largest frequency shifts observed for the t46c - cn 
   probe occurred across the two most important steps in the catalytic 
   cycle : ( 1 ) upon the conversion between the model michaelis 
   menten 
   e : nadp : fol complex and the initial e : nadp : thf 
   product complex ( 4.1 cm ) , which is analogous 
   to the hydride transfer step ; ( 2 ) across the enzyme turnover step , 
   which is the release of thf ( 5.5 cm ) . for the 
   l54c - cn probe , 
   the most significant change occurred upon the formation 
   of the ternary e : nadp : fol complex ( 2.4 cm ) . 
   crystal structures of the wt enzyme have shown that the t46 and 
   l54 residues retain their positions and orientations in the five catalytic 
   complexes , suggesting that the inserted cn probes likely experience 
   limited freedom of movement in the active site . 
   md simulations confirm that there are no major conformational 
   changes in the probe orientation during 5 ns trajectories . 
   therefore , 
   the observed changes in the microenvironments as the system moves 
   through the various complexes in the catalytic cycle are likely due 
   to protein conformational rearrangements that induce changes in the 
   electrostatic interactions between the probes and their surroundings . 
   note also that the redox state of the cofactor ( nadph / nadp ) has very little effect on both the ir and c nmr data 
   ( table s4 ) . 
   in many cases , we also 
   observed changes in the peak width [ full - width 
   at half - maximum ( fwhm ) ; table s4 ] , which 
   usually reflects the variation of chemical environments sampled by 
   the probe as well as the strength of intermolecular interactions such 
   as hydrogen bonding . 
   for example , the l54c - cn probe , which is located 
   in the folate binding site , reported peak widths that are approximately 
   two times smaller in complexes with bound fol or thf . 
   this observation 
   is consistent with the probe sampling a less fluctuating and more 
   well - defined electrostatic environment resulting from the various 
   interactions that organize the fol / thf to bind in the active site . 
   there is also some evidence indicating that the e : nadph complex and 
   the apoenzyme exhibit a greater degree of hydrogen bonding between 
   a solvent water molecule and the l54c - cn probe . 
   one indication of 
   this phenomenon is obtained from inspection of the c 
   nmr - ir relationship , which shows that the data for the above two complexes 
   deviate from the rest of the data in a manner that implicates greater 
   specific solvent hydrogen - bonding interactions ( figure s4 ) . 
   md simulations support the presence of additional 
   solvent hydrogen - bonding interactions in these two complexes for the 
   l54c - cn probe , with a water molecule hydrogen bonded to the nitrile 
   probe in nearly half of the configurations . 
   we also observed a slightly 
   broader ir peak in the e : nadph : fol complex , which can be attributed 
   to the slow reduction of fol over the course of the ir measurement . 
   this is not unexpected since the l54c - cn probe is located in the folate 
   binding pocket . 
   these trends are less obvious for the t46c - cn probe , 
   which seems to be located in a more fluctuating environment , as indicated 
   by the relative ir peak widths . 
   we also prepared the isotopically 
   enriched cys - cn 
   variants ( t46c - cn and l54c - cn ) to obtain 
   the complementary c nmr data for all of the studied complexes . 
   while the magnitude of the observed changes are different because 
   the c nmr chemical shift is less sensitive to specific 
   solvent hydrogen - bonding interactions than is the cn vibrational stretching 
   frequency , the c nmr data illustrate 
   similar trends to those shown by the corresponding ir data . 
   in particular , 
   we observed a changing microenvironment around the scn 
   probes as the enzyme evolves through the catalytic cycle ( figure 2b ) . 
   similar to the ir data , the microenvironment 
   detected at the t46c - cn probe exhibits larger changes 
   along the catalytic cycle than that at the l54c - cn probe . 
   this trend is expected due to the proximity of the t46c - cn site to the regions of the substrates that undergo the hydride 
   transfer reaction . 
   overall , the experimental data summarized as figure 2 clearly demonstrate a dynamic electrostatic environment 
   in the active site of ecdhfr across its catalytic 
   cycle . 
   the differences in the microenvironments of the two probes 
   and the way they change among the different complexes , especially 
   between the closed and occluded conformations , in the ecdhfr catalytic cycle also provide an indication of the highly heterogeneous 
   nature of the enzyme active site . to identify 
   the various environmental contributions that give rise to the observed 
   cn stretching vibrational frequency shifts along the catalytic cycle , 
   we conducted qm / mm simulations to monitor the electrostatic interactions 
   experienced by the probes for the complexes along the catalytic cycle . 
   these data provide information about how the microenvironments of 
   the probes reorganize across individual steps of the catalytic cycle . 
   we simulated the ir spectra of the t46c - cn and l54c - cn mutants of ecdhfr for two different complexes along the catalytic cycle : 
   ( 1 ) the ternary complex with the reactant analog fol and nadp bound , which mimics the e : nadph : dhf state prior to hydride 
   transfer ; ( 2 ) the ternary complex with the product thf and nadp bound , which is the e : nadp : thf state following 
   hydride transfer . 
   first we examined the local environment around the 
   t46c - cn probe , which is located near the midpoint between the two 
   bound ligands ( figure 1a ) . 
   the qm region includes 
   the t46c - cn residue , the substrate ( either fol or thf ) , and the nearest 
   water molecule to the nitrile nitrogen , as depicted in figure 3a . 
   the qm region was determined by calculating the 
   cn frequency including the entire probe microenvironment in the qm 
   region for a subset of the configurations sampled and comparing these 
   results to those obtained with smaller qm regions . 
   the details of 
   the procedure used to determine the qm region are provided in the supporting information . 
   configurations from md 
   simulations for the ( a ) t46c - cn and ( b ) 
   l54c - cn ecdhfr mutants . 
   the residues with thicker 
   lines are included in the qm region for the qm / mm calculations of 
   vibrational frequencies . 
   figure 4a presents the calculated 
   and experimentally 
   measured ir spectra for the t46c - cn mutant for both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . 
   the calculated 
   ir spectrum for the closed conformation ( solid black curve ) has a 
   maximum at 2165.8 cm , which is within 1.8 cm of the measured peak in the experiments . 
   the agreement 
   between the calculated and experimental absolute frequencies is potentially 
   fortuitous , but the level of agreement is similar to that calculated 
   in our previous studies of ksi . 
   the fwhm 
   of the calculated ir spectrum for the closed conformation is 11 cm , which is in good agreement with the experimentally 
   measured line width of 14 cm . 
   the calculated ir 
   spectrum for the occluded product state ( solid red curve ) has a maximum 
   at 2162.1 cm . 
   the calculated shift between these 
   two states is 3.7 cm , which is in excellent 
   agreement with the experimentally measured shift of 4.1 cm . 
   note that our md simulations and cn frequency calculations 
   are performed on the nanosecond time scale . 
   the probe could experience 
   multiple additional conformations on the longer time scale associated 
   with the ir measurements . during our md simulations of 5 ns , no major 
   structural rearrangements of the probe orientation occurred , indicating 
   that we are only sampling a single conformational state . since the 
   starting configurations for our simulations were based on crystal 
   structures that include the probe , we expect to be sampling the most 
   stable and therefore the dominant configuration of the probe . 
   moreover , 
   the agreement between the calculated and experimental cn frequency 
   shifts provides further validation of the methodology . 
   calculated and experimentally 
   measured ir spectra of the cn vibrational 
   stretching frequency for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound ( closed conformation ; 
   black line ) and with nadp / thf bound ( occluded conformation ; 
   red line ) . 
   the solid lines are the simulated spectra from the qm / mm 
   calculations , and the dotted lines are the measured spectra from the 
   ftir experiments . 
   in addition to the ir 
   spectra , we calculated the electric field 
   at the midpoint of the cn bond for configurations sampled by the md 
   trajectory using the methodology described above . from these calculations , 
   we can correlate the measured vibrational frequency shifts to changes 
   in electrostatics from the protein environment . 
   figure 5a depicts the calculated electric field along the cn axis 
   for the two states . 
   the average projected electric field for the closed 
   ( nadp / fol ) and occluded ( nadp / thf ) states 
   are 15.9 and 19.0 mv / cm , respectively . using the experimental 
   stark tuning rate of 0.7 cm/(mv / cm ) in conjunction 
   with eq 1 , the difference of 3.1 mv / cm 
   corresponds to a shift in the vibrational frequency of 2.2 
   cm . 
   the difference between this frequency shift 
   arising from only classical electrostatics and the actual shift of 
   ca . 
   4.0 cm obtained from both calculations 
   and experiments is attributed to the quantum mechanical aspects of 
   hydrogen - bonding interactions and polarization effects that are not 
   included in the linear relation given by eq 1 . 
   calculated electric field along the cn bond at the midpoint of 
   this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr 
   systems with nadp / fol bound ( closed conformation ; black 
   line ) and with nadp / thf bound ( occluded conformation ; 
   red line ) . previously it has been shown experimentally and computationally that significant deviations of 
   10 cm from the vibrational frequency shifts 
   predicted by eq 1 can occur when the nitrile 
   probe participates in specific hydrogen - bonding interactions . according 
   to our md simulations , 
   the t46c - cn thiocyanate probe is solvent accessible 
   in both the closed and occluded conformations and directly hydrogen 
   bonds to a water molecule , as depicted in figure 3a . in both protein conformations , 
   the nitrile probe is also 
   able to act as a hydrogen - bond acceptor from the amine group in the 
   substrate ( fol / thf ) backbone . 
   note that the observation of hydrogen - bonding 
   interactions with the probe for these two states is consistent with 
   the experimental ir - nmr correlation data in figure 
   s4 . 
   thus , the difference between the frequency shift 
   of 2.2 
   cm predicted from eq 1 , 
   which includes only classical electrostatic effects , and the shift 
   of 3.7 cm obtained from the qm / mm calculations 
   of the full system can be attributed to hydrogen - bonding interactions 
   with the water molecule and polarization effects from the water molecule 
   and the substrate ( fol / thf ) . to confirm this hypothesis , we performed 
   the qm / mm frequency analysis with only the probe residue ( t46c - cn ) 
   in the qm region ( i.e. , switching the water molecule and the substrate 
   from the qm region to the mm region ) . in this case , the calculated 
   frequency shift between the two states was 2.6 cm , which agrees well with the 2.2 cm shift 
   obtained from eq 1 . 
   beyond the calculation 
   of the total electric field at the probes , 
   we can decompose the field along the cn axis into contributions from 
   individual residues in the enzymatic complexes . 
   in essence , this methodology 
   allows us to decompose the microenvironment surrounding the cn probe 
   into the constituent parts , which then allows us to identify the major 
   contributing components . for the t46c - cn probe ( figure 3a ) , the sum of the contributions from the main contributors 
   given in table 1 corresponds to a change in 
   electric field along the cn axis of 2.9 mv / cm , which is consistent 
   with the change of 3.1 mv / cm calculated with the entire enzyme . 
   the contributions from the residues in the closed conformation 
   are shown pictorially in figure 6a , where the 
   residues that are colored blue contribute positively and those that 
   are colored red contribute negatively to the total field along the 
   cn bond . 
   for the t46c - cn probe , the nadp cofactor and 
   the nearest water molecule are the dominant contributors to the change 
   in the electric field along the cn axis , although asn18 and ser49 
   also make significant contributions ( table 1 ) . 
   the presence of a positive charge on the nicotinamide ring and 
   the short 4  distance between the nitrile nitrogen and 
   the aromatic nitrogen in the nicotinamide ring lead to a strong contribution 
   to the electric field in the closed conformation . after the transition 
   to the occluded conformation 
   , the cofactor is excluded from the active 
   site , and the magnitude of the contribution to the field from the 
   nicotinamide ring is much smaller due to the larger distance of 14 
    between the two nitrogen atoms . 
   this effect of the movement 
   of nadp on the electric field at the probe demonstrates 
   how atomic motions can significantly alter the local electrostatics 
   in the active site . 
   moreover , the 3.2 mv / cm change in the 
   electric field contributed by asn18 and ser49 is consistent with the 
   structural rearrangement from the closed to the occluded conformation . 
   specifically , the amide nitrogen of asn18 is 1.5  closer 
   to the nitrile probe in the closed conformation than in the occluded 
   conformation , and the hydroxyl group of ser49 is 1.0  
   closer to the nitrile probe in the closed conformation . 
   contributions 
   to the calculated electric field along the cn bond 
   at the midpoint of this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound and the met20 
   loop in the closed conformation . 
   the color for each residue corresponds 
   to the calculated values in tables 1 and 2 , respectively , using the color scale provided , 
   although residues contributing a magnitude > 4.0 mv / cm are depicted 
   in the darkest color . 
   the residues that contribute negatively to the 
   field are colored red , the residues that contribute positively to 
   the field are colored blue , and the residues that have no net contribution 
   to the field along the cn bond are colored white . 
   changes 
   in the electric field along 
   the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . 
   additionally , following the closed 
   to occluded transition , the 
   cn probe becomes more accessible to the solvent . 
   the contribution 
   to the electric field along the cn axis from the closest water molecule 
   changes by 7.46 mv / cm during this transition . 
   moreover , the 
   sign of the average electric field along the cn axis due to this water 
   molecule is different for the closed and occluded conformations , with 
   a value of 2.14 mv / cm for the closed conformation and 5.32 
   mv / cm for the occluded conformation . 
   analysis of the md trajectories 
   indicates that a water molecule is hydrogen bonded to the nitrile 
   probe 26% of the time in the closed ( nadp / fol ) 
   conformation and 52% of the time in the occluded ( nadp / thf ) conformation . 
   the positive contribution 
   to the electric field along the cn axis from the nearest water molecule 
   in the closed conformation is due to configurations in which the nearest 
   water molecule has its negatively charged oxygen atom pointing toward 
   the nitrile . 
   these configurations are stabilized by the hydrogen - bonding 
   interaction of the water molecule with the hydroxyl group of ser49 , 
   as shown in figure s5 . 
   the negative contribution 
   to the field in the occluded conformation arises from configurations 
   in which a hydrogen atom of the nearest water molecule is oriented 
   toward the probe , forming a hydrogen bond between the water molecule 
   and the cn group . 
   thus , this computational approach is also able to 
   probe the detailed active site hydration status , which is not easily 
   accessible with experimental means . 
   we performed the same analysis for the l54c - cn mutant 
   to examine the microenvironment near the folate binding pocket . 
   figure 4b depicts the calculated and experimentally measured 
   vibrational spectra for the l54c - cn mutant in both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . 
   the l54c - cn 
   probe is distal from the active site , and it is located in a relatively 
   hydrophobic pocket near the carboxylate tail of the substrate , as 
   shown in figure 1 . 
   the qm region used for this 
   mutant includes the l54c - cn residue , phe31 , and arg57 , as depicted 
   in figure 3b . 
   the qm region was determined 
   by calculating the cn frequency including the entire probe microenvironment 
   in the qm region for a subset of the configurations sampled and comparing 
   these results to those obtained with smaller qm regions . 
   the details 
   of the procedure used to determine the qm region are provided in the supporting information . 
   the calculated frequency 
   shift between the two states for the l54c - cn probe is 2.5 
   cm , which is in reasonable agreement with the 
   shift of 1.0 cm measured experimentally . 
   figure 5b depicts the calculated electric 
   fields along the cn bond for the l54c - cn mutant in the two states . 
   the calculated average electric fields along the cn probe in the closed 
   and occluded conformations are 8.4 and 13.8 mv / cm , 
   respectively . using the experimental stark tuning rate of 0.7 cm/(mv / cm ) in conjunction with eq 1 
   , this shift of 5.4 mv / cm corresponds to a shift in vibrational 
   frequency of 3.8 cm due to only classical 
   electrostatic interactions . to model the situation with only classical 
   electrostatic interactions , we calculated the shift in the vibrational 
   frequency when only the residue containing the thiocyanate group ( l54c - cn ) 
   is treated quantum mechanically and the remainder of the system , including 
   phe31 and arg57 , 
   the calculated 
   frequency shift when only the probe residue is treated quantum mechanically 
   is 3.5 cm , which is consistent with the 
   value of 3.8 cm obtained from eq 1 . 
   however , this frequency shift is greater than the 
   shift of 2.5 cm calculated with the larger 
   qm region . 
   the greater shift predicted by the purely classical electrostatic 
   model indicates that the polarization effects due to the phe31 and 
   arg57 residues decrease the vibrational frequency shift relative to 
   that predicted by classical electrostatics . 
   table 2 provides the contribution to the 
   electric field along the cn axis for several key residues near the 
   l54c - cn probe . 
   the sum of these contributions is 7.3 mv / cm , 
   which is slightly higher than the change of 5.4 mv / cm calculated 
   with the entire enzyme , indicating that other residues are also contributing 
   to this field . in a similar manner to the analysis for the t46c - cn 
   mutant , 
   the arg57 residue exhibits the largest change 
   between the two states with a net shift of 6.58 mv / cm . in 
   the closed conformation , 
   the line connecting the midpoint of the cn 
   bond to the protonation site of the positively charged arg57 is nearly 
   perpendicular to the cn axis , resulting in almost zero contribution 
   to the electric field along the cn axis . 
   the change in the electric 
   field contribution by arg57 is likely due to a geometrical shift that 
   moved the positive charge on arg57 away from this orthogonal orientation 
   with respect to the probe in the occluded conformation . 
   interestingly , 
   while the substrate exerts a large contribution to the electric field 
   along the cn axis , this contribution remains nearly constant between 
   the two states . 
   the stable orientation of the cn axis relative to 
   the negatively charged carboxylate moiety in the bound substrate results 
   in negligible change in the contribution of the substrate to the electric 
   field at the cn probe . 
   changes 
   in the electric field along 
   the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . while the calculated electric 
   fields along the cn axes of the probes provide a measure of the electrostatic 
   microenvironments within the active site , the electric field along 
   the hydride transfer d 
   encouraged by the good agreement between the experimental 
   and the simulated data described above , we applied the same methodology 
   to evaluate the total electric field at the midpoint of the d  a 
   axis and the field projected along the d  a axis in the closed 
   ( e : nadp : fol ) state . 
   the negative value of the field indicates 
   that it points from the substrate to the cofactor and that this field 
   will facilitate the transfer of a negatively charged hydride from 
   the cofactor to the substrate . in other words , the projection and 
   the magnitude of the field along the d  a axis in the ternary 
   michaelis complex is highly favorable for the hydride transfer reaction 
   to occur . 
   the substrate and cofactor contribute a total of 32.4 
   mv / cm to the field along the d  a axis . 
   the rest of the protein 
   contributes approximately an additional 31% ( 15.4 
   mv / cm ) of the total field . 
   also , the solvent molecules and ions contribute 
   an additional 1.1 mv / cm to the field along the d  a 
   axis . 
   this analysis suggests that a major function of the enzyme 
   is to bring the substrate and cofactor into a configuration that realizes 
   the highly favorable field for facilitating the hydride transfer reaction 
   ( figure 7 ) . in the absence of the enzyme 
   , it 
   would be less probable for the reacting species , nadph and dhf , to 
   achieve the same relative orientation in solution . 
   moreover , this 
   analysis provides a new perspective on the role of electrostatic catalytic 
   contributions in dhfr . 
   it is currently impossible to experimentally 
   measure the electric field along the d  a axis , but the calculations 
   provide reasonable estimates . a favorable electric field of 15.4 
   mv / cm from the protein and 32.4 mv / cm from the ligands along 
   the d  a axis 
   could potentially reduce the free energy barrier 
   by several kcal / mol . while these values 
   suggest a substantial catalytic contribution generated from the active 
   site electrostatic environment , it is important to acknowledge that 
   the full mechanism of catalysis is far more complex than a simplistic 
   evaluation based solely on the electric field along the d  a 
   axis . 
   depiction of the component of the electric field along the hydride 
   transfer d  a axis calculated from an md simulation of wt ecdhfr with nadp / fol bound and the met20 loop 
   in the closed conformation . 
   the color for each residue corresponds 
   to the calculated electric field values using the color scale provided , 
   although residues contributing a magnitude > 4.0 mv / cm are depicted 
   in the darkest color . 
   the three arrows in the red oval represent the 
   total electric field of 48.9 mv / cm ( purple ) , the field of 
   32.4 mv / cm resulting from the ligands ( yellow ) , and the field 
   of 16.5 mv / cm resulting from the rest of the system ( green ) 
   projected along the d  a axis . 
   we also decomposed this field into the contributions from 
   all of 
   the protein residues , and our analysis identified many residues that 
   contribute to the electric field along the d  a axis . 
   figure 7 depicts the contributions from each of the residues 
   to the field along the d  a axis . 
   the residues that are colored 
   blue contribute positively and those that are colored red contribute 
   negatively to the field pointing from the substrate to the cofactor 
   along the d  a axis . 
   the residues that are shown with side chains 
   contribute to the field with a magnitude > 1 mv / cm . 
   interestingly , there is a strong degree of overlap between the 
   residues that strongly contribute to the field along the d  a 
   axis and the network of coupled motions that has been identified for 
   dhfr catalysis . 
   the residues ile14 , tyr100 , 
   and asp122 contribute 1.2 , 4.2 , and 2.4 mv / cm , 
   respectively , and were all implicated in the network of coupled motions . 
   additional residues that strongly contribute to the electric field 
   along the d  a axis are lys32 ( 3.2 mv / cm ) , arg52 ( 3.9 
   mv / cm ) , and arg57 ( 4.2 mv / cm ) . 
   the residues that contribute 
   strongly against the field along the d  a axis include asp27 
   ( 5.4 mv / cm ) , arg98 ( 3.4 mv / cm ) , and his124 ( 3.0 mv / cm ) . 
   all of these 
   residues represent potential targets to alter the electrostatic contribution 
   to catalysis . 
   we investigated 
   the changing electrostatic environment at two different 
   thiocyanate probes inserted into the active site of ecdhfr for five complexes along the catalytic cycle . 
   our results illustrate 
   a changing electrostatic environment near the hydride transfer site 
   ( t46c - cn ) as well as a smaller change in key regions of the folate 
   binding pocket ( l54c - cn ) . 
   the changes in electrostatic environment 
   are associated with conformational changes occurring along the catalytic 
   cycle of ecdhfr . 
   experimental evidence for the changing 
   electrostatic environment includes both ir frequency and c nmr measurements , which interrogate the electric field along the 
   cn axis and the total electric field at the probe . 
   these experimental 
   data demonstrate fundamental aspects of the electrostatic landscape 
   for catalysis in ecdhfr . 
   molecular dynamics 
   simulations provided an atomic - level picture 
   of the local environment experienced by the thiocyanate probes in 
   complexes that model the states prior to and subsequent to the hydride 
   transfer reaction . 
   the qm / mm vibrational frequency calculations reproduced 
   the experimentally measured frequency shifts for these two states , 
   which span the closed to occluded transition associated with hydride 
   transfer . 
   analysis of the md trajectories provided insight into the 
   conformational changes occurring between these two states . the factors 
   leading to the vibrational frequency shifts were broken down into 
   classical electrostatic effects , specific hydrogen - bonding interactions , 
   and polarization effects . 
   thus , the vibrational frequency shifts are 
   not simply a manifestation of the classical electric fields but rather 
   include additional contributions from the environment . 
   moreover , the 
   electric fields along the cn axis of the probes were decomposed into 
   contributions from specific residues , ligands , and solvent molecules . 
   the electric field along the hydride d  a axis was also calculated 
   with the experimentally validated methodology . 
   the calculations illustrate 
   that the cofactor and substrate , as well as the enzyme , impose a substantial 
   electric field along the d  a axis that facilitates hydride 
   transfer . 
   overall , experimental and theoretical data provide 
   evidence for 
   significant electrostatic changes in the microenvironments that constitute 
   the heterogeneous ecdhfr active site as the enzyme 
   progresses along the catalytic cycle . 
   the electrostatic interactions 
   between the protein and the ligands assist in orienting the reacting 
   species in a geometry that maintains a large electric field favoring 
   hydride transfer from the cofactor to the substrate . 
   the enzyme active 
   site environment also provides a significant portion ( 1/3 ) 
   of the total electric field that facilitates the hydride transfer 
   reaction , suggesting the catalytic importance of enzymatic conformational 
   changes that alter the active site electrostatics . 
   future work will 
   investigate the five trapped states along the catalytic cycle for 
   the two probes studied in this work as well as additional probe sites 
   in the ecdhfr system to provide a comprehensive picture 
   of the electrostatic landscape for the entire catalytic cycle .
summary: electrostatic interactions play an 
   important role in enzyme catalysis 
   by guiding ligand binding and facilitating chemical reactions . 
   these 
   electrostatic interactions are modulated by conformational changes 
   occurring over the catalytic cycle . 
   herein , the changes in active 
   site electrostatic microenvironments are examined for all enzyme complexes 
   along the catalytic cycle of escherichia coli dihydrofolate reductase ( ecdhfr ) by incorporation 
   of thiocyanate probes at two site - specific locations in the active 
   site . the electrostatics and degree of hydration of the microenvironments 
   surrounding the probes 
   are investigated with spectroscopic techniques 
   and mixed quantum mechanical / molecular mechanical ( qm / mm ) calculations . 
   changes in the electrostatic microenvironments along the catalytic 
   environment lead to different nitrile ( cn ) vibrational stretching 
   frequencies and 13c nmr chemical shifts . 
   these environmental 
   changes arise from protein conformational rearrangements during catalysis . 
   the qm / mm calculations reproduce the experimentally measured vibrational 
   frequency shifts of the thiocyanate probes across the catalyzed hydride 
   transfer step , which spans the closed and occluded conformations of 
   the enzyme . 
   analysis of the molecular dynamics trajectories provides 
   insight into the conformational changes occurring between these two 
   states and the resulting changes in classical electrostatics and specific 
   hydrogen - bonding interactions . 
   the electric fields along the cn axes 
   of the probes are decomposed into contributions from specific residues , 
   ligands , and solvent molecules that make up the microenvironments 
   around the probes . 
   moreover , calculation of the electric field along 
   the hydride donor  acceptor axis , along with decomposition of 
   this field into specific contributions , indicates that the cofactor 
   and substrate , as well as the enzyme , impose a substantial electric 
   field that facilitates hydride transfer . 
   overall , experimental and 
   theoretical data provide evidence for significant electrostatic changes 
   in the active site microenvironments due to conformational motion 
   occurring over the catalytic cycle of ecdhfr .