Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 Å) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity. To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 Å) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity. Hyperthermophilic organisms grow at temperatures above 80 °C. Proteins from these organisms are themselves optimally active between 60 and 125 °C and serve as paradigms for the characterization of factors responsible for protein fold stability and flexibility. Hyperthermophilic enzymes have also attracted considerable attention because of a range of biotechnological applications (1Adams M.W. Perler F.B. Kelly R.M. Biotechnology (NY). 1995; 13: 662-668Crossref PubMed Scopus (189) Google Scholar, 2Adams M.W. Kelly R.M. Trends Biotechnol. 1998; 16: 329-332Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Sequence comparison studies and structural analyses carried out on hyperthermophilic proteins and their mesophilic homologues have shown that thermal stability is associated with multiple factors, including an increase in hydrogen bonding, complex salt bridge formation, and helix stabilization by acidic residues. The commonly accepted hypothesis is that increased thermal stability is due to enhanced conformational rigidity of the molecular structure (3Jaenicke R. Naturwissenschaften. 1996; 83: 544-554Crossref PubMed Scopus (19) Google Scholar). Hyperthermophilic enzymes are also characterized by a higher temperature of maximum activity (3Jaenicke R. Naturwissenschaften. 1996; 83: 544-554Crossref PubMed Scopus (19) Google Scholar, 4Zavodszky P. Kardos J. Svingor G.A. Petsko G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7406-7411Crossref PubMed Scopus (493) Google Scholar). The more rigid hyperthermophilic enzyme would then require higher temperatures to achieve the requisite conformational flexibility for activity. Experiments have shown that thermostable enzymes exhibit reduced structural flexibility at room temperature with respect to their mesophilic homologues (4Zavodszky P. Kardos J. Svingor G.A. Petsko G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7406-7411Crossref PubMed Scopus (493) Google Scholar, 5Wrba A. Schweiger A. Schultes V. Jaenicke R. Zavodsky P. Biochemistry. 1990; 29: 7584-7592Crossref PubMed Scopus (187) Google Scholar), whereas others, on α-amylase (6Fitter J. Heberle J. Biophys. J. 2000; 79: 1629-1636Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and on rubredoxin (7Hernandez G. Jenney Jr., F.E. Adams M.W. LeMaster D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3166-3170Crossref PubMed Scopus (169) Google Scholar, 8Grottesi A. Ceruso M.A. Colosimo A. Di Nola A. Proteins. 2002; 46: 287-294Crossref PubMed Scopus (61) Google Scholar), have shown the opposite effect, i.e. the thermostable homologues were found to be more flexible, suggesting stabilization through entropic effects. Relations between flexibility and stability are, therefore, complex. Atomic fluctuations only were measured in these experiments and interpreted in terms of flexibility. It is important to point out that reduced structural “flexibility” does not necessarily imply a more “rigid” structure. Atoms are maintained in a structure by forces that link them to their neighbors. In terms of a force field, the width of the potential well in which an atom moves is a measure of its flexibility in terms of a root mean square fluctuation amplitude (<u2>), whereas the detailed shape of the well reflects the rigidity of the structure, in terms of an effective force constant (<k>). In this picture, the stability would be given by the depth of the well (9Tehei M. Madern D. Pfister C. Zaccai G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14356-14361Crossref PubMed Scopus (93) Google Scholar). Two types of potential wells are illustrated in Fig. 1. In the case of harmonic motion (Fig. 1A), flexibility and rigidity are related, as expected intuitively. The potential is given by V(u)=12<k>u2, and the atomic mean square fluctuation is related to the force constant by <u2(T)>= kBT/<k> (10Bicout D.J. Zaccai G. Biophys. J. 2001; 80: 1115-1123Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 11Zaccai G. Science. 2000; 288: 1604-1607Crossref PubMed Scopus (624) Google Scholar); a less rigid harmonic structure is indeed more flexible. Protein structures, however, are not harmonic at physiological temperature, and atoms move in different types of potential. Fig. 1B illustrates a simplified, extreme case in which an atom can move quite freely in a “box” formed by its neighbors but cannot go out of the box. Mathematically this can be described by the square well potential shown. The flexibility, in this case, is a temperature-independent constant value, while the effective force constant stopping the atom from leaving the box is infinitely high. Flexibility and rigidity are independent parameters therefore that, as shown below, can be obtained separately from neutron scattering data. We proposed a novel neutron scattering approach that provides independent measurements of the atomic mean fluctuations in a protein structure (the global thermal flexibility) and of the mean force constant maintaining the structure, which we called resilience because “rigidity” has been used in a broadly qualitative way (11Zaccai G. Science. 2000; 288: 1604-1607Crossref PubMed Scopus (624) Google Scholar, 12Zaccai G. Tehei M. Scherbakova I. Serdyuk I. Gerez C. Pfister C. J. Phys. IV. 2000; 10Google Scholar). In the present work, the combined analysis of neutron data on dynamics, on the one hand, with activity and stability data, on the other, for hyperthermophilic and mesophilic enzymes of the malate lactate dehydrogenase family revealed a strong adaptation of resilience and mean square fluctuations to physiological temperature. By performing comparative analysis using the sequences and the three-dimensional crystal structures, we suggested mechanisms that govern the high thermal stability of Methanococcus jannaschii malate dehydrogenase (Mj MalDH) 3The abbreviations used are: Mj MalDHMethanococcus jannaschii malate dehydrogenaseOc LDHOryctolagus cunniculus lactate dehydrogenaseN/mNewtons/meterSs LDHSus scrofa LDH. through increased resilience. Methanococcus jannaschii malate dehydrogenase Oryctolagus cunniculus lactate dehydrogenase Newtons/meter Sus scrofa LDH. Oryctolagus cunniculus lactate dehydrogenase (Oc LDH) was from Sigma. Mj MalDH was prepared as described in Ref. 13Madern D. Mol. Microbiol. 2000; 37: 1515-1520Crossref PubMed Scopus (16) Google Scholar. Activity—The measurements of the rate of NADH oxidation at 340 nm were carried out in a 1.00-cm thermostated cuvette containing 0.2 mm NADH and 0.6 mm appropriate substrate buffered with 50 mm Tris-HCl, pH 8. Pyruvate and oxaloacetate were used with Oc LDH and Mj MalDH, respectively. The optimal activity was expressed as the percentage of the maximal activity relative for each enzyme. Stability—Samples of Oc LDH and Mj MalDH were incubated in 50 mm Tris-HCl, pH 8, for 30 min at the specified temperature, and their residual activities were measured. The samples were covered with paraffin oil to avoid evaporation at high temperature. The guanidinium hydrochloride-induced unfolding of Oc LDH and Mj MalDH was monitored using circular dichroism spectroscopy. Samples (0.5 mg/ml) were incubated in 50 mm Tris-HCl, pH 8, for 24 h at the specified GdnHCl concentration. Far-UV circular dichroism spectra were recorded between 190 and 260 nm with an interval of 1 nm and an integration time of 15 s. Neutron Scattering Experiments—Samples for neutron scattering were concentrated protein solutions (∼200 mg/ml) in 20 mm KCl, 20 mm Tris-HCl, pH 7.5 buffer. Experiments were performed on the back-scattering spectrometer IN13 at the Institut Laue Langevin, Grenoble, France (information on the institute and the instrument is available at www.ill.fr). The incident neutron wavelength used was λ = 2.23 Å. Neutron spectrometers are characterized by their energy resolution, Δω, and scattering vector, Q, range, corresponding to time and space windows related to 1/Δω and 1/Q, respectively. Elastic incoherent scattering data were collected with an energy resolution of 8 μeV in a scattering vector range of 1.2 Å-1≤Q ≤2.2 Å-1, corresponding to a space-time measurement window of ∼1 Å in 0.1 ns. Samples were contained in aluminum sample holders with a 0.3-mm path length. Correction for multiple scattering was not performed because the transmission of all samples was ∼90%, indicating that this effect can be neglected. Corrections for self absorption using Paalman-Pings coefficients were carried out with standard programs. All samples, including the vanadium and empty aluminum can, were oriented at 135° with respect to the incident neutron beam direction. The scattering of the buffer alone was much lower than the protein solution scattering, barely above the scattering of the aluminum container. It (the scattering of the buffer) was subtracted from the data with no correction for protein-excluded volume. The data were normalized by the vanadium scattering to correct for detector response. In the instrument space-time window and according to a Gaussian approximation, the incoherent elastic scattered intensity can be analyzed as (14Smith J.C. Q Rev. Biophys. 1991; 24: 227-291Crossref PubMed Scopus (307) Google Scholar) I (Q, 0±Δω) = constant·exp{(1/6) (-<u2> Q2)}, where Q is 4πsinθ/λ, 2θ is the scattering angle, and λ the incident neutron wavelength; <u2> values include all contributions to motions in the accessible space and time windows from vibrational fluctuations (usually expressed as a Debye-Waller factor) as well as from diffusional motions. The validity of the Gaussian approximation for the mean square fluctuation <u2> and its analogy to the Guinier formalism for small angle scattering by particles in solution has been discussed by Réat et al. (15Réat V. Zaccai G. Ferrand M. Pfister C. Cusack S. Büttner H. Ferrand M. Langan P. Timmins P. Biological Macromolecular Dynamics. Adenine Press, Schenectady, NY1997: 117-122Google Scholar) and more recently by Gabel (16Gabel F. Eur. Biophys. J. 2005; 34: 1-12Crossref PubMed Scopus (43) Google Scholar). In the Guinier formalism a radius of gyration Rg2 of particles in solution is calculated (17Guinier A. Fournet G. Small Angle Scattering of X-rays. John Wiley & Sons, New York, London1955Google Scholar). The particle equivalent is the volume swept out by a single proton during the time scale of the experiment (∼100 ps). The analogy holds if the motion is localized well within the space-time window defined by the Q and energy transfer ranges, respectively. The Guinier approximation is valid if Rg2*Q2≈1. Following our definition of <u2>, Rg2=1/2*<u2>. As a consequence, the Gaussian approximation is valid in the domain where <u2>*Q2≈2. The mean square fluctuations <u2> at a given temperature T were calculated according to the Gaussian approximation as (Fig. 2): Ln [I (Q, 0±Δω)] = constant + A* Q2. The mean square fluctuations were therefore calculated as: <u2> = -6 A. The <u2> values were then plotted as a function of absolute temperature T (Fig. 3). The value of the root mean square fluctuation <u2> in absolute Å units quantifies the global flexibility of the system studied. An effective mean force constant <k′>, defining mean resilience, can be calculated from the derivative of <u2> plotted versus temperature T (10Bicout D.J. Zaccai G. Biophys. J. 2001; 80: 1115-1123Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 11Zaccai G. Science. 2000; 288: 1604-1607Crossref PubMed Scopus (624) Google Scholar) (Fig. 3): <k′>= 0.00276/(d <u2>/dT). The numerical constants allow the expression of <k′> in N/m with <u2> in Å2 and T in Kelvin. The mean square fluctuations <u2>, the effective force constants <k′>, and their respective errors were calculated from the slopes of weighted straight line fits using the Marquart-Levenberg algorithm. Sequence and comparison was performed using the J. D.J. PubMed Scopus Google Scholar). The structural was performed using the 1996; PubMed Scopus Google Scholar) and with C. Trends Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). were using the J. Mol. PubMed Scopus Google Scholar). analysis was performed using D.J. J. Mol. PubMed Scopus Google Scholar) with of Å. and hydrogen were calculated and using C. PubMed Scopus Google Scholar). protein volume and were calculated with the PubMed Scopus Google Scholar). radius of Å was used for structure was calculated using C. Trends Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). dehydrogenase from the Mj MalDH was with its mesophilic homologue, the lactate dehydrogenase from O. cunniculus (Oc Mj MalDH is a of the family of malate which are and have similar to the LDH D. Mol. Microbiol. 2000; 37: 1515-1520Crossref PubMed Scopus (16) Google Scholar, C. J. Mol. 2001; PubMed Scopus Google Scholar, D. J. Mol. 2002; PubMed Scopus Google Scholar). and activities of the enzymes were measured as a function of temperature (Fig. The temperatures of optimal activity are °C for Oc LDH and °C for Mj The relative of the enzymes were by residual activity measurements and using guanidinium as (Fig. and The residual activity is in Oc LDH at °C and in Mj MalDH above °C. The guanidinium unfolding for Oc LDH and Mj MalDH are and respectively. these data that the hyperthermophilic protein is more Macromolecular have been to explore protein adaptation to high temperature. (4Zavodszky P. Kardos J. Svingor G.A. Petsko G.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7406-7411Crossref PubMed Scopus (493) Google Scholar), J. S. Biochemistry. PubMed Scopus Google Scholar), I. C. C. Proteins. 2000; PubMed Scopus Google Scholar), and neutron scattering J. R. A. Biochemistry. 2001; PubMed Scopus Google Scholar) are the The flexibility of protein is in conformational of the to In during and the of the of protein can be by experiments a measure of protein experiments not protein and be performed in which the the and the of proteins (9Tehei M. Madern D. Pfister C. Zaccai G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14356-14361Crossref PubMed Scopus (93) Google Scholar, F. Madern D. Zaccai G. J. Mol. PubMed Scopus Google Scholar). experiments measure structural flexibility and for protein require the of a for for the of In however, different have a different to the and the is only to structural the of in The of the protein by and provides in terms of order parameters and is an between the time of neutron scattering experiments and of Neutron scattering experiments can be performed to physiological on proteins of and not require can be used to explore specific effects. The provides on atomic fluctuation in a given time scale in absolute Neutron scattering experiments were performed on the IN13 spectrometer at the Institut Laue We that the space-time window of the experiments to ∼1 Å in 0.1 ns. the as the of Å in 0.1 for not to the scattering that experiments be performed in to physiological that of the neutron scattering experiments were performed in In this space-time atoms the motions of the and atom to which are and on protein F. D. U. Tehei M. M. Zaccai G. Q Rev. Biophys. 2002; PubMed Scopus Google Scholar). The mean square fluctuation <u2> of the sample atoms was measured as a function of temperature. Two parameters on were the value of the root mean square fluctuation <u2> in absolute Å which quantifies the global flexibility of the and the mean resilience <k′> in absolute N/m calculated from the of <u2> versus T (11Zaccai G. Science. 2000; 288: 1604-1607Crossref PubMed Scopus (624) Google Scholar). The global mean square fluctuations <u2> in the proteins measured by neutron scattering as a function of temperature are shown in Fig. In the temperature range, the <u2> of the hyperthermophilic protein above of its mesophilic is in with neutron studies on that fluctuation for the protein (6Fitter J. Heberle J. Biophys. J. 2000; 79: 1629-1636Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). It not be however, that this higher flexibility a lower rigidity of the mean square fluctuation at °C for Mj MalDH, the temperature of its optimal was obtained by at corresponding to a root mean square fluctuation of 1.5 Å. value for Mj MalDH is to that for Oc LDH at °C. The that the enzymes have conformational flexibility to the temperature in with the hypothesis that adaptation of proteins to different physiological temperatures to enzymes in characterized by similar conformational flexibility R. Eur. J. 1991; PubMed Scopus Google Scholar). The mean resilience values of the protein were calculated as effective mean force constants from the slopes of <u2> versus T in Fig. a more protein structure and The mean resilience is an order of magnitude for Mj MalDH than for Oc LDH (0.15 we that the resilience of MalDH from measured in solution is N/m (9Tehei M. Madern D. Pfister C. Zaccai G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14356-14361Crossref PubMed Scopus (93) Google Scholar). The resilience of as a is N/m and to N/m at physiological temperature (11Zaccai G. Science. 2000; 288: 1604-1607Crossref PubMed Scopus (624) Google Scholar). The higher stability of Mj MalDH therefore, with resilience higher than the resilience of mesophilic are the and structural mechanisms that govern the higher stability and resilience of Mj Sequence and with Mj MalDH enzyme is of and which is by than the corresponding found in the Oc LDH. The of of the of in Mj MalDH is with that found in Oc LDH The of and in Mj MalDH and are much than the corresponding and found in Oc LDH. has been between adaptation to high temperature and the and percentage in protein C. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of is and in Mj MalDH and Oc respectively. The value is therefore much higher for Mj MalDH than for Oc LDH The strong between the effective resilience and the percentage a as to which protein stabilization forces are by The increase of the value for the Mj MalDH, with Oc is due to an increase in that hydrogen D. C. Zaccai G. 2000; PubMed Scopus Google Scholar), and and therefore to a of the contributions to the energy The three-dimensional crystal structure of the enzyme Oc LDH is not to the structural of the between resilience and adaptation to high temperature, we a mesophilic of Oc LDH structure has been dehydrogenase from Sus scrofa LDH) to the domain and has the physiological temperature and of as Oc LDH. temperature also is the as that of Oc LDH. the two proteins a strong of Mj MalDH, LDH and Oc temperature of activity from this temperature of temperature of temperature of of and Mj and and Mj and from this from Ref. from this used for structural Mj MalDH root mean square temperature of and Mj and from this from Ref. M. R. 1995; Full Text Full Text PDF PubMed Scopus Google root mean square in a The structural of the LDH with Mj MalDH structure and Fig. a high of of the three-dimensional The volume by the Mj MalDH is than that by the of LDH. in can be obtained also by a in the and volume of M. P. A. S. A. R. H. J. Mol. 2002; PubMed Scopus Google Scholar). Two are present in the Mj MalDH and a volume of The and the volume of of Mj MalDH are than that present in the LDH are due to a of Rev. PubMed Scopus Google Scholar). by can increase the of proteins M. PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google Scholar), and the to in the rigid of a protein structure Biochemistry. PubMed Scopus Google Scholar). The of the to which atoms on the protein can with The of Mj MalDH is than that of LDH The has in that is related to The of each square of a in energy of C. PubMed Scopus Google Scholar). is an of the for Mj MalDH, which the resilience through a of the of small of contributions to the energy comparison MalDH structure performed with volume of volume of of hydrogen structure of hydrogen in a The value is much higher for Mj MalDH than for LDH The are to and hydrogen have been proposed to a in the of enzyme stability at high temperature Full Text Full Text PDF PubMed Scopus Google Scholar, M. R. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The of in Mj MalDH is increased with its mesophilic the proposed effect of the of are on the protein with the is also an important for Neutron scattering experiments on the malate dehydrogenase from that the enzyme is more where is more (9Tehei M. Madern D. Pfister C. Zaccai G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14356-14361Crossref PubMed Scopus (93) Google Scholar). The of potential hydrogen is higher for Mj MalDH than that of the equivalent of LDH Mj MalDH has than its mesophilic In Mj MalDH, of the are in and in The with LDH is small but as the helix and of the mesophilic homologues are and respectively. the of to structure in Mj MalDH is higher than is in LDH The increase in structure to a in the of the and to an increase in the of hydrogen the of hydrogen is higher for Mj MalDH We however, that the higher resolution of the LDH structure Å with Å for Mj MalDH to the of more hydrogen suggesting the be All these Mj MalDH structural to a global increase in protein resilience, through and hydrogen contributions to the energy the between protein stability and enzyme activity is in a corresponding between resilience and flexibility. The structural of stability in the malate dehydrogenase have been discussed M. P. A. S. A. R. H. J. Mol. 2002; PubMed Scopus Google Scholar, A. Madern D. Zaccai G. A. G. R. J. Mol. PubMed Scopus Google Scholar). stability from a of different that factors, as increased value of protein and increased as well as a of and volume of increased and increased hydrogen are responsible for a more protein and to an increase in protein resilience, suggesting the of contributions to the energy in the proteins in which entropic are a less be more

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