Cite this: ACS Catal. Article Views Altmetric -. Citations 7. Abstract High Resolution Image. Detailed knowledge of the mechanisms employed by enzymes to speed up chemical processes can serve as a guide for the design of novel biocatalysts. Comparative studies of dihydrofolate reductase DHFR homologues offer a unique opportunity to elucidate the impact of protein dimerization on the chemical step. H 4 F is the reduced form of folic acid and is the primary carrier of C1 units in many important cellular processes.
It is the precursor of derivatives that participate in a myriad of biosynthetic reactions. Scheme 1. High Resolution Image. DHFRs of different organisms share important parts of their sequences and most of the residues of their active site are identical.
Although dimerization has been proposed as a strategy for enhancing enzymatic thermostability, its effect on catalysis has not been systematically explored for DHFRs.
The lower catalytic power of enzymes with reduced flexibility such as TmDHFR has been proposed to support a possible link between protein dynamics and enzyme catalysis. A combination of experimental and computational approaches has been used to clarify different aspects of biocatalysis including the relationship between catalysis and enzyme structure and flexibility.
Kinetic isotope effects KIEs can be measured experimentally and predicted by computer simulations; they are one of the most efficient techniques to shed light onto the intimate aspects of enzymatic catalysis. Isotopic substitution slows protein motions due to the mass increase, whereas within the Born—Oppenheimer approximation 28 the electronic potential energy surface remains unperturbed.
Experimental measurements on enzymatic KIEs have been carried out for several protein systems such as purine nucleoside phosphorylase, 26 HIV protease, 32 alanine racemase, 33 pentaerythritol tetranitrate reductase, 34 and several dihydrofolate reductases DHFRs. Here, we report a theoretical investigation of the effect of protein motions on hydride transfer catalyzed by TmDHFR. We have also computed enzymatic KIEs and compared our computational results with those obtained from previously published experimental work.
The calculation of the rate constant of the chemical step was based on transition state theory TST with the inclusion of tunneling contributions and recrossing effects as a preexponential factor. The initial coordinates were taken from the X-ray crystal structure of T. Water molecules within 2. Five sodium cations were added in order to neutralize the total charge of the system and their initial position was selected according to the electrostatic potential created by the protein.
The ratio between the masses of the simulated heavy and light enzymes was 1. The quantum subsystem contained 76 atoms, including parts of the cofactor nicotinamide ring and ribose and the substrate pteridine ring and the N -methylene-substituted p -aminobenzoyl, pABA of chain A.
The quantum atoms were described by the AM1 Hamiltonian, 56 modified using specific reaction parameters denoted as AM1-SRP developed previously for DHFR by Major and co-workers that provide excellent activation free energies and geometries. Periodic boundary conditions were employed combined with the minimum image convention in all the simulations. The umbrella sampling approach was used 60 with the system restrained to remain close to the desired values of the reaction coordinate by means of the addition of a harmonic potential with a force constant of kJ mol —1 A —2 , which allows good overlap between windows.
The reaction coordinate was explored in a range from — 2. The probability distributions obtained from MD simulations within each individual window were combined by means of the weighted histogram analysis method WHAM. This procedure was repeated for 11 temperatures ranging from to K. The classical mechanical activation free energies presented in Table 1 were obtained as the average of three different PMFs for each of the simulated temperatures , , and K Figure S1.
For other temperatures, the classical mechanical activation free energy was obtained from just one PMF. Table 1. All the terms in eqs 1 — 3 see Methods , obtained at , , , and K, are listed in Table 1. Results obtained at other temperatures , , , , , , and K are provided in the Supporting Information see Tables S2 and S3. The results at K are presented in Table 2 see Table S4 for results also at and K and show that the values for the free energy barriers of the monomeric and dimeric forms are identical with the difference of about 0.
As mentioned before, previous theoretical studies predicted significantly smaller rate constants for the monomeric form 18 which is in disagreement with later experimental observations. Our results confirm that dimerization itself does not alter the catalytic efficiency at room temperature or the contribution of tunneling.
Instead, dimerization rigidifies some structural elements of the protein the loops involved in the dimer interface, as discussed below , which keeps the structural integrity of the protein at higher temperatures and shifts the activation enthalpy—entropy balance to a less negative activation entropy and a larger activation enthalpy. Although the number of points used in the fit is small, the observation of an increased value for the activation entropy in the monomer with respect to the dimer is also supported by other observations see below.
This effect of dimerization on the activation entropy—enthalpy balance results in an activation free energy for the dimeric TmDHFR that increases less steeply with temperature, providing in this way larger catalytic rate constants at higher temperatures.
This is clearly a beneficial effect of dimerization for thermophilic enzymes. Table 2. This feature, also observed by Warshel and co-workers, 14 can be explained because the open conformation observed for the M20 loop in TmDHFR allows more water molecules to enter the active site. Water molecules stabilize the RS better than the TS because the charge on the substrate is more localized.
This is reflected in the position of the first peak of the radial distribution function that is shifted from 5. In general, water molecules present in active sites can follow the electronic changes in the chemical system but at the cost of a larger reorganization energy, which is then translated into a larger activation free energy and a lower reaction rate for the chemical reaction. The closed conformation prevents the formation of some interactions between the residues of the M20 loop and the substrate or the cofactor that could stabilize the TS, as deduced from the comparison of the intermolecular distances reported in Tables S5 and S6.
This contributes to the reduction in the catalytic efficiency of the enzyme by increasing the activation enthalpy due to the lack of some favorable interactions.
The enthalpy increase is partly compensated by the fact that as less interactions are established between the substrate and the enzyme the protein is less stiffened when passing from RS to TS, contributing to a less negative activation entropy, as determined in the previous section. Although dimerization itself does not significantly alter the rate of hydride transfer at room temperature, it is nevertheless important for the thermal stability of TmDHFR and thus indirectly maintains its catalytic activity at higher temperatures.
Both experimental measurements 6 and the simulations presented here Table 1 reveal that the activation free energy of TmDHFR increases less steeply with temperature than for other dihydrofolate reductase catalyzed reactions.
An increase of the temperature is translated into larger fluctuations of these loops that could eventually lead to denaturation of the monomeric form at larger temperatures.
This increased flexibility with the temperature is dramatically attenuated in the dimer. Thus, our simulations suggest that dimerization makes the whole protein structure more rigid, not only the active site, and is thus more resistant to melting which is in agreement with the experimental results.
Analysis of cross-correlation motions between residues for the dimeric and monomeric forms of TmDHFR see Figure S5 reveals a subtle mechanism for the coupling between the two monomers in the dimer. Dimerization introduces new correlations between motifs of the different subunits.
In particular, strong positive correlations are observed between loops FG and GH belonging to different subunits. This is not unexpected because these loops are located in the dimerization area.
Interestingly, the motions of these loops are correlated with the catalytically important M20 loops. The M20 loops of the two subunits also display positive cross-correlations between them. The coupling observed between the motions of the loops of both subunits could be at the origin of the thermal stability of the dimer observed in Figure 4. In particular, analysis of the backbone dihedral angles of the M20 loop shows that this loop can adopt different conformations in the monomeric and dimeric forms of TmDHFR see Figure S6.
Within our approach, the reaction coordinate depends exclusively on the degrees of freedom of the chemical system the substrate and the cofactor and hence any nonequilibrium effects of protein dynamics should be captured in this transmission coefficient. The temperature dependence of both experimental and theoretical EKIEs shows that they are close to unity at the optimum working temperature of each DHFR enzyme see Figure 5.
All enzymes have been optimized by evolution to work at their physiological temperatures. This means that their three-dimensional structure is preorganized to catalyze the chemical reaction, requiring a minimal amount of reorganization, that is, motions in the enzymatic structure, to reach the TS at the temperature where the host organism thrives. If the participation of protein motions is minimized, the change from a light version of an enzyme to the heavy counterpart has minimal effects on the reaction rate constants and the EKIEs are close to unity.
If the working temperature is changed, then different conformational states can become accessible to the enzyme. These conformations will not have been optimized at nonphysiological temperatures, so that increased dynamics is required to reach the TS, which in turn will increase the impact of the protein mass on the reaction rate constant.
In this regard, it is interesting to compare the different behaviors of two thermophilic enzymes, namely BsDHFR a moderately thermophilic monomeric enzyme and TmDHFR a hyperthermophilic dimeric enzyme Figure 5.
Thermal adaptation in BsDHFR is achieved by removing themolabile residues and extending secondary structural elements, which provokes some elements to become significantly more flexible than in the mesophilic EcDHFR. The most flexible motifs such as the loops are locked in particular conformations at the dimer interface and the structural integrity of the enzyme is preserved.
As a result, the participation of protein motions is minimized over the whole range of temperatures studied and the EKIEs are always close to unity. These are responsible of the environmental reorganization taking place during the chemical step and are not affected by the change of the mass of the protein. Their contribution to the reorganization of the active site during hydride transfer remains unaltered when going from light to the heavy TmDHFR.
Interestingly, an in silico estimation of the solvent KIE as the ratio between the transmission coefficient obtained in deuterated and normal water, gives a value of 1. The results presented here show that dimerization is not responsible for the reduced catalytic efficiency of TmDHFR; the reduced efficiency is likely to be a consequence of changes introduced in the primary sequence of TmDHFR that stabilize the dimer.
These water molecules stabilize RS relative to TS and this is reflected in a larger change in the hydride donor—acceptor distance when going from the RS to the TS.
This effect contributes to the increase in the free energy barrier of the reaction. The presence of water molecules also helps diminish the absolute value of the activation entropy, a property of TmDHFR that makes it more catalytically competent at high temperatures. This reduction is more evident at higher temperatures, where thermal fluctuations can lead to unfolding of the enzyme. However, some protein flexibility is a prerequisite for the reaction so that the active site can accommodate the charge redistribution on the path to the TS.
No significant changes are observed when comparing protein—substrate distances at RS and TS at different temperatures; this means that the pattern of protein interactions with the chemical system, despite not being as optimal as in other DHFRs, are already present in the Michaelis complex.
Analysis of the temperature dependence of both experimental and theoretical EKIEs obtained for other DHFRs shows that a value close to unity is reached at their respective physiological temperatures; the values of the enzyme KIEs increase when moving toward nonphysiological temperatures.
In the hyperthermophilic TmDHFR on the other hand, EKIEs remain close to unity over the whole temperature range, indicating a minor participation of protein motions during barrier crossing. Supporting Information. Author Information. Rudolf K. The authors declare no competing financial interest. Nature , , — , DOI: Nature Publishing Group. A review. There are possible amino acid sequences for a residue protein, of which the natural evolutionary process has sampled only an infinitesimal subset.
De novo protein design explores the full sequence space, guided by the phys. Computational methodol. Almost all protein engineering so far has involved the modification of naturally occurring proteins; it should now be possible to design new functional proteins from the ground up to tackle current challenges in biomedicine and nanotechnol.
Macmillan Magazines. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temp. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small mols. We have now investigated whether hydrogen tunnelling occurs at elevated temps. Using a thermophilic alc. Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temp.
These findings provide exptl. American Chemical Society. The authors have compared the global flexibility of the mesophile and the thermophile at their physiol. The main observation from this set of expts. The results reported herein, taken together with the similar nature of hydrogen transfer at the enzymes' optimum temps.
Henzler-Wildman, Katherine A. Jordan; Karplus, Martin; Kern, Dorothee. The synergy between structure and dynamics is essential to the function of biol. Thermally driven mol. However, very little is understood about the connection of these functionally relevant, collective movements with local at. Here, the authors show that pico- to nano-second timescale at. The fast, local mobilities differed between mesophilic and hyperthermophilic AKs, but were strikingly similar at temps.
The connection between different timescales and the corresponding amplitudes of motions in AK and their linkage to catalytic function is likely to be a general characteristic of protein energy landscapes.
In contrast to all other enzyme reactions investigated previously, including DHFR from Escherichia coli EcDHFR , for which isotopic substitution led to decreased reactivity, the rate const. Clearly, dynamical coupling is not a universal phenomenon that affects the efficiency of enzyme catalysis. Biochemistry , 49 , — , DOI: Dimerization is believed to play a key role in the high thermal stability of TmDHFR, which is reflected in a melting temp. The dimer interface of TmDHFR is composed of a hydrophobic core with charged residues around the periphery.
In particular, Lys of each subunit forms three-membered salt bridges with Glu and Glu of the other subunit. Our results indicate that these salt bridges are key for the high thermal stability of TmDHFR but are not a requirement for dimerization. Dimer dissociation during the nano-electrospray process should result in largely folded monomeric species, which are formed from dimeric species carrying 22 or more charges. Normal electrospray conditions result in dimer dissociation Fig. The low pH 1.
Assignment of the distinct charge state distributions to differentiate unfolded species is supported by CD and fluorescence experiments, which suggest that non-native, partially unfolded states are predominantly populated at low pH. Although an identification of the specific protonation sites is not possible from mass spectrometric data, a comparison may be made with the environments of the sites in the crystal structures.
Figure 14 a — c shows the distribution of the Arg, Lys and His residues which are potential sites for protonation. Of the eight Arg residues which have high pKa values, as many as seven are involved in interactions with neighbouring, negatively charged residues in the crystal structures Fig. Similarly, 6 out of 22 lysine residues are involved in interactions with neighbouring negatively charged residues not shown.
In some cases, ion pair interactions are mediated through bridging water molecules Fig. Interestingly, only one of the five His residues participates in electrostatic interactions Fig.
Assuming that the memory of solution phase structures is maintained in the gas phase, under conditions where a dimeric state is populated, it is possible that these water molecules are mass spectrometrically detectable. Water-mediated ionic interactions in Pf TIM. There are 19 Glu, 14 Asp and a C-terminal carboxylic acid, which are potentially ionisable.
The detection of clearly asymmetric charge state distributions for the monomeric species suggests that largely folded and partially folded states of isolated monomeric subunits are populated in the gas phase. Hydration is an inevitable consequence of the milieu in which proteins exist in cells and may have an important functional role Meyer, The crystal structures of proteins have provided a great deal of detailed structural information on water molecules which are bound to protein surfaces Bottoms et al.
The presence of a very large number of water molecules in the crystal lattice permits observation of not only strongly bound solvent molecules, but also allows visualisation of water networks which hold proteins together in the solid state.
In the case of multimeric proteins and protein—protein complexes, water molecules often act as bridges between protruding side chains of amino acid residues on the interacting surfaces Paliwal et al.
Although the strength of interaction between the individual water molecules and protein residues is hard to estimate, crystallographic observations may be used to identify tightly bound and invariant water molecules in protein structures, determined from differentially hydrated crystals Nagendra et al.
Simplistically, the water molecules which show multiple interactions to proteins are likely to be bound more strongly to the macromolecular surface. Notably in the case of Pf TIM, the number of water molecules detected which are bound to the diverse charge states under different conditions corresponds closely to the number of highly conserved water molecules, which are identified in the crystal structures see preceding section.
In the dimer structure, two invariant water molecules are found in almost all structures. Interestingly, species corresponding to two bound water molecules are observed in all the charge states of the monomeric protein.
It is relevant to note that preferential hydration of specific charge states has been observed in BPTI Woenckhaus et al.
HOH and HOH are conserved in most of the crystal structures examined and mediate interaction between positively and negatively charged sites Fig. It is likely that such waters may indeed be strongly bound to the protein even in the gas phase. Hydrogen bonds which are formed between ionic and neutral species are estimated to be exceptionally strong Meot-Ner, and the stability will presumably be enhanced in a solvent-free, low dielectric environment.
Further information on gas phase hydration may be obtained by comparisons between wild-type and mutant protein structures, where specific hydration sites are eliminated by suitable mutation. We thank Prof. Jayant B. Google Scholar. Oxford University Press is a department of the University of Oxford.
It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Experimental methods. Water molecules in the Pf TIM crystal structure. Detection of the protein dimers, multiple monomeric states and hydrated forms of Plasmodium falciparum triosephosphate isomerase in the gas phase.
Thakur , Suman S. Oxford Academic. Mousumi Banerjee. Balaram 3. E-mail: pb mbu. Revision received:. Cite Cite Suman S. Select Format Select format. Permissions Icon Permissions. Pace, C. Protein ionizable groups: pK values and their contribution to protein stability and solubility.
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NMR 24 , — Guntert, P. Brunger, A. Acta Crystallogr. D Biol. Version 1. DeLano, W. Download references. You can also search for this author in PubMed Google Scholar. All authors discussed the results and edited the manuscript. Correspondence to Nina Kronqvist. Reprints and Permissions. Kronqvist, N. Nat Commun 5, Download citation. Received : 01 October Accepted : 14 January Published : 10 February Anyone you share the following link with will be able to read this content:.
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If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Molecular biology Structural biology. Abstract The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved.
You have full access to this article via your institution. Introduction Spider dragline silk, one of the toughest biomaterials known, is produced through the assembly of large proteins spidroins with remarkable biochemical properties. Figure 1: Comparison of dimeric wt NT structures.
Full size image. Figure 2: Dimerization analysis and stability measurements of NT mutants. Figure 7: Proposed mechanism for NT dimerization. Discussion The molecular mechanism now proposed involves a three-step process in which NT monomers first associate Fig. Protein expression and purification The plasmids were heat-shock-transformed into chemically competent E.
Tryptophan fluorescence measurements Fluorescence emission spectra were measured on a spectrofluorometer Tecan Safire 2 using Costar black polystyrene assay plates with 96 flat bottom wells.
Even if conditions during thermal alteration in the absence of air are far different from those used in frying, techniques applied have been of great help in the identification of nonpolar dimers in used frying fats. The levels found indicate that nonpolar dimers might be one of the most relevant groups among the alteration compounds [3]. The structure of polar dimers is still largely unknown. Difficulties are due to the heterogeneity in this group of compounds.
Firstly, different oxygenated functions are likely to be present in oxidized monomers before dimer formation, or generated by the oxidation of nonpolar dimers. Secondly, more than one functional group can be present in the same dimeric molecule. Lastly, the oxygen may or may not be involved in the dimeric linkage. Therefore, the large number of possible combinations results in a complex mixture that is difficult to separate. Under these circumstances, studies have paid more attention to defining the composition of alteration products than to the mechanisms involved in dimer formation.
Among the studies carried out, those giving information on the dimers found in fats and oils used in frying or heated under simulated frying conditions are especially interesting.
After separating fractions of different polarity by adsorption chromatography, identification techniques, including mass spectrometry, nuclear magnetic resonance and infrared spectroscopy, have been helpful in providing evidence of some of the dimeric structures formed.
The following results are noteworthy:. In summary, because of the high number of nonoxygenated and oxygenated dimeric FAME found in fats and oils subjected to frying conditions, it is difficult to obtain more detailed analyses of these compounds.
The difficulty increases even more when dealing with the underivatized TAG. Definite structures for compounds with molecular weight higher than dimers have not been reported, neither as methyl esters from frying fats nor in model systems. This is not strange considering that much more research remains to be done on structure elucidation and quantification of simpler molecules, i. Moreover, the potential number of different structures in trimer formation increases exponentially with respect to those compounds of lower molecular weight as many different dimeric structures may be combined with many different monomeric structures.
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