Microcalorimetric determination of displacement adsorption enthalpies of protein refolding on a moderately hydrophobic surface at 308 K

Both microcalorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of guanidine – denatured lysozyme (Lys) refolding on the surface of hydrophobic interaction chromatography (HIC) packings at 308 K were carried out and compared with that at 298 K. Study shows that both temperature and concentration of guanidine hydrochloride (GuHCl) affect the molecular mechanism of hydrophobic interaction of protein with adsorbent based on the analysis of dividing ΔH values into three kinds of enthalpy fractions. The adsorption in higher concentrations of GuHCl (>1.3 mol L^–1) at 308 K is an enthalpy-driving process, and the adsorption under other GuHCl concentrations is an entropy-driving process. The fact that the Lys denatured by 1.8 mol L^–1 GuHCl forms a relatively stable intermediate state under the studied conditions will not be changed by temperature.     

人工设计逆醛缩酶RA95.5-8F催化β-羟基酮C—C裂解的理论研究

采用量子力学与分子力学组合(QM/MM)方法对人工设计逆醛缩酶RA95.5-8F催化β-羟基酮化合物裂解反应的机理进行了研究. 结果表明, 裂解反应主要包括赖氨酸Lys1083对底物的亲核进攻、 Schiff碱形成、 烯胺水解及C—N断裂等过程, C—N键裂解生成丙酮为整个反应的决速步骤, 能垒为106.27 kJ/mol; 活性中心的赖氨酸Lys1083、 酪氨酸Tyr1051、 天冬酰胺Asn1110和酪氨酸Tyr1180构成一个催化四联体, Lys1083通过与底物形成席夫碱对底物进行活化, Tyr1051作为催化酸碱参与质子转移过程, 催化四联体的氢键网络有利于反应过渡态的稳定并使R-构型的底物更容易结合在活性位点, 导致RA95.5-8F对R构型底物具有高的选择性和催化活性.     

The amyloidogenicity of gelsolin is controlled by proteolysis and pH.

BACKGROUND: Normally, gelsolin functions in plasma as part of the actin-scavenging system to assemble and disassemble actin filaments. The Asp 187-->Asn (D187N) Asp 187-->Tyr (D187Y) gelsolin mutations facilitate two proteolytic cuts in the parent protein generating a 71-residue fragment that forms amyloid fibrils in humans, putatively causing Finnish type familial amyloidosis (FAF). We investigated the role of the D187N mutation in amyloidogenicity using biophysical studies in vitro. RESULTS: Both the recombinant wild-type and D187N FAF-associated gelsolin fragments adopt an ensemble of largely unfolded structures that do not self-associate into amyloid at pH 7. 5. Incubation of either fragment at low pHs (6.0-4.0) leads to the formation of well-defined fibrils within 72 hours, however. CONCLUSIONS: The D187N mutation has been suggested to destabilize the structure of the gelsolin parent protein (specifically domain 2), facilitating two proteolytic cleavage events. Our studies demonstrate that generating the largely unstructured peptide is not sufficient alone for amyloid formation in vitro (on a time scale of months). A drop in pH or an analogous environmental change appears necessary to convert the unstructured fragment into amyloid fibrils, probably through an associative mechanism. The wild-type gelsolin fragment will make amyloid fibrils from pH 6 to 4 in vitro, but neither the wild-type fragment nor fibrils have been observed in vivo. It is possible that domain 2 of wild-type gelsolin is stable in the context of the whole protein and not susceptible to the proteolytic degradation that affords the 71-residue FAF-associated peptide.     

Spin-trapping of the p-benzyne intermediates from ten-membered enediyne calicheamicin gamma1I

In the presence of thiols, the ten-membered-ring enediyne calicheamicin gamma1I generates a p-benzyne biradical that initiates oxidative cleavage of double-stranded DNA. Application of spin-trapping has successfully provided ESR and mass spectroscopic evidence for the formation of the monoadducts with phenyl tert-butyl nitrone (PBN). [reaction: see text].     

Translational diffusion constants of the amino acids: measurement by NMR and their use in modeling the transport of peptides

In this work, the translational self-diffusion constants, DT's, of 12 amino acids (Ala, Arg, Asn, Asp, Cys, Glu, His, Ile, Lys, Met, Phe, and Ser) are measured by field gradient NMR and extrapolated to infinite dilution. The experiments were carried out in D2O at 298 K at pD approximately =3.5 in 50 mM sodium phosphate buffer. Of these 12 amino acids, 6 are being reported for the first time (Asp, Cys, Glu, His, Lys, and Met) and the remaining 6 (Ala, Arg, Asn, Ile, Phe, and Ser) are compared with DT's from the literature. When corrected for differences in solvent viscosity and temperature, the discrepancy between DT's measured in the present work and those reported previously is always <8%, which is reasonable given the range of values reported previously by different groups. With the present work, DT's for all of the amino acids are now available. These diffusion constants are then used in modeling studies of the diffusion and free solution electrophoretic mobility, mu, of several model peptides. For this set of peptides, it is shown that modeling using revised input parameters results in improved agreement between model and experimental mobilities.     

Gamma2-, gamma3-, and gamma(2,3,4)-amino acids, coupling to gamma-hexapeptides: CD spectra, NMR solution and X-ray crystal structures of gamma-peptides

There are numerous possible gamma-amino acids with different degrees of substitution and with various constitutions and configurations. Of these the gamma4- and the like- and unlike-gamma(2,4)-amino acids have been previously used as building blocks in gamma-peptides. The synthesis of gamma2-, gamma3-, and gamma(2,3,4)-peptides is now described. The corresponding amino acids have been prepared by Michael addition of chiral N-acyl-oxazolidinone enolates to nitro-olefins, with subsequent reduction of the NO2 to NH2 groups. Such additions to E-2-methyl-nitropropene provide (2R,3R,4R)-2-alkyl-3-methyl-4-amino-pentanoic acid derivatives (9, 10, 11). Stepwise coupling and fragment coupling lead to gamma-di-, tri-, and hexapeptides (12-23), which were fully characterized. The crystal structures of one of the gamma-amino acids (2,3-dimethyl-4-amino-pentanoic acid x HCl, 9a), of a gamma(2,3,4)-di- and a gamma(2,3,4)-tetrapeptide (20, 22) are described, and the NMR solution structure in MeOH of a gamma(2,3,4)-hexapeptide (3) has been determined (using TOCSY, COSY, HSOC, HMBC and ROESY measurements and a molecular dynamics simulated-annealing protocol). A linear conformation (sheet-like), a novel (M) helix built of nine-membered hydrogen-bonded rings, and (M) 2.6(14) helices have thus been identified. NMR measurements at different temperatures (298-393 K) and H/D-exchange rates obtained for the gamma(2,3,4)-hexapeptide are interpreted as evidence for the stability of the 2.6(14) helix (no "melting") and for its non-cooperative folding mechanism. CD Spectra of the gamma-peptides have been measured in MeOH and CH3CN, indicating that only the protected and unprotected gamma(2,3,4)-hexapeptide is present as the 2.6(14) helix in solution. The structures of the gamma2- and gamma3-hexapeptides (1, 2) could not be determined.     

Stereospecific capillary electrophoresis assays using pentapeptide substrates for the study of Aspergillus nidulans methionine sulfoxide reductase A and mutant enzymes

Stereospecific capillary electrophoresis‐based methods for the analysis of methionine sulfoxide [Met(O)]‐containing pentapeptides were developed in order to investigate the reduction of Met(O)‐containing peptide substrates by recombinant Aspergillus nidulans methionine sulfoxide reductase A (MsrA) as well as enzymes carrying mutations in position Glu99 and Asp134. The separation of the diastereomers of the N‐acetylated, C‐terminally 2,4‐dinitrophenyl (Dnp)‐labeled pentapeptides ac‐Lys‐Phe‐Met(O)‐Lys‐Lys‐Dnp, ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp and ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp was achieved in 50 mM Tris‐HCl buffers containing sulfated β‐CD in fused‐silica capillaries, while the diastereomer separation of ac‐Lys‐Asp‐Met(O)‐Asp‐Lys‐Dnp was achieved by sulfated β‐CD‐mediated MEKC. The methods were validated with regard to range, linearity, accuracy, limits of detection and quantitation as well as precision. Subsequently, the substrates were incubated with wild‐type MsrA and three mutants in the presence of dithiothreitol as reductant. Wild‐type MsrA displayed the highest activity towards all substrates compared to the mutants. Substitution of Glu99 by Gln resulted in the mutant with the lowest activity towards all substrates except for ac‐Lys‐Asn‐Met(O)‐Asp‐Lys‐Dnp, while replacement Asn for Asp134 lead to a higher activity towards ac‐Lys‐Asp‐Met(O)‐Asn‐Lys‐Dnp compared with the Glu99 mutant. The mutant with Glu instead of Asp134 was the most active among the mutant enzymes. Molecular modeling indicated that the conserved Glu99 residue is buried in the Met‐S‐(O) groove, which might contribute to the correct placing of substrates and, consequently, to the catalytic activity of MsrA, while Asp134 did not form hydrogen bonds with the substrates but only within the enzyme.     

The impact of protonation and deprotonation of 3-methyl-2'-deoxyadenosine on N-glycosidic bond cleavage

The enzyme-substrate contacts that are believed to be involved in depurination by proton transfer have been modelled by protonation and deprotonation of 3-methyl-2'-deoxyadenosine (3-MDA) using quantum mechanical calculations in the gas-phase and solution media. The change in the charge distribution on the sugar ring and nucleobase that is introduced by the protonation and deprotonation strongly affects the N-glycosidic bond length. The unimolecular cleavage and hydrolysis of the N-glycosidic bond, involving D(N)*A(N) and A(N)D(N) pathways, have been considered at several levels of theory. The trend in the energy barriers is A(N)D(N) > cleavage > D(N)*A(N). All probable proton transfer reactions resulting from enzyme-substrate contacts do not facilitate the N-glycosidic bond cleavage of 3-MDA. The deprotonation of 3-MDA that may result from the interaction between H6 and enzyme do not facilitate bond cleavage. The protonation at N7 induces more positive charge on the sugar ring and further facilitates the depurination relative to the protonation at N1. The changes in the charges calculated on the ribose and nucleobase are in good relationship with the C1'-C2', C1'-O4', and N-glycosidic bond lengths along the cleavage. The change in energy barrier ΔE of glycosidic bond cleavage from the gas-phase to solution media strongly depends on the charge of the species.     

Peptidomic approach based on combined capillary isoelectric focusing and mass spectrometry for the characterization of the plasmin primary products from bovine and water buffalo beta-casein

The main peptides produced by hydrolysis of water buffalo beta-casein with plasmin were characterized by capillary electrophoresis and mass spectrometry and compared with their bovine homologous. A novel breakdown product arising from the hydrolysis of water buffalo beta-casein, originated by the presence of a plasmin-sensitive Lys bond at position 68 was identified, which was not present in bovine beta-casein. On the basis of this evidence, an improved procedure for the detection and the differentiation of the products of plasmin hydrolysis of bovine and water buffalo beta-casein by capillary isoelectric focusing was set-up. In the experimental conditions, the gamma-casein from the two species was efficiently separated. Comparison of the capillary electropherograms with those obtained by ultra-thin-layer isoelectric focusing, the reference method for routine analysis of plasmin digests of casein, suggests that capillary electrophoresis isoelectric focusing may constitute a successful alternative to the traditional slab gel electrophoresis analysis of plasmin digests of casein either for basic structural studies or for applications in the quality assessment of dairy products.     

Alteration of the formate dehydrogenase isoelectric point by rational design

In order to extend the pH stability optimum for NAD⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Pseudomonas sp. 101 (PseFDH), four mutant enzymes with Lys112Pro, Lys231Ala, Lys231Ser, and Lys317Asn substitutions were obtained by site-directed mutagenesis. The choice of the mutation sites and the types of substituting amino acids were based on the alignment of amino acid sequences of FDHs from various sources and the analysis of the three-dimensional structure of PseFDH. The kinetic properties and temperature stability were studied for all obtained mutant forms. It is shown that the substitutions in positions 112 and 231 slightly improved the kinetic properties; meanwhile, the Lys317Asn mutant possessed a decreased affinity for the coenzyme. A thermal stability assay for the obtained mutants revealed that the substitutions in positions 112 and 231 result in just a slight destabilization of the enzyme, while Lys317Asn substitution causes a significant decrease in thermal stability. The isoelectric point was decreased by 0.1 points for all obtained mutant forms.     

Cytochrome c552 mutants: structure and dynamics at the active site probed by multidimensional NMR and vibration echo spectroscopy

Spectrally resolved infrared stimulated vibrational echo experiments are used to measure the vibrational dephasing of a CO ligand bound to the heme cofactor in two mutated forms of the cytochrome c552 from Hydrogenobacter thermophilus. The first mutant (Ht-M61A) is characterized by a single mutation of Met61 to an Ala (Ht-M61A), while the second variant is doubly modified to have Gln64 replaced by an Asn in addition to the M61A mutation (Ht-M61A/Q64N). Multidimensional NMR experiments determined that the geometry of residue 64 in the two mutants is consistent with a non-hydrogen-bonding and hydrogen-bonding interaction with the CO ligand for Ht-M61A and Ht-M61A/Q64N, respectively. The vibrational echo experiments reveal that the shortest time scale vibrational dephasing of the CO is faster in the Ht-M61A/Q64N mutant than that in Ht-M61A. Longer time scale dynamics, measured as spectral diffusion, are unchanged by the Q64N modification. Frequency-frequency correlation functions (FFCFs) of the CO are extracted from the vibrational echo data to confirm that the dynamical difference induced by the Q64N mutation is primarily an increase in the fast (hundreds of femtoseconds) frequency fluctuations, while the slower (tens of picoseconds) dynamics are nearly unaffected. We conclude that the faster dynamics in Ht-M61A/Q64N are due to the location of Asn64, which is a hydrogen bond donor, above the heme-bound CO. A similar difference in CO ligand dynamics has been observed in the comparison of the CO derivative of myoglobin (MbCO) and its H64V variant, which is caused by the difference in axial residue interactions with the CO ligand. The results suggest a general trend for rapid ligand vibrational dynamics in the presence of a hydrogen bond donor.     

A dinuclear Ni(I) system having a diradical Ni2N2 diamond core resting state: synthetic, structural, spectroscopic elucidation, and reductive bond splitting reactions

One-electron reduction of the square-planar nickel precursor (PNP)NiCl ( 1) (PNP (-) = N[2-P(CHMe 2) 2-4-methylphenyl] 2) with KC 8 effects ligand reorganization of the pincer ligand to assemble a Ni(I) dimer, [Ni(mu 2-PNP)] 2 ( 2), containing a Ni 2N 2 core structure, as inferred by its solid-state X-ray structure. Solution magnetization measurements are consistent with a paramagnetic Ni(I) system likely undergoing a monomer <--> dimer equilibrium. The room-temperature and 4 K solid-state X-band electron paramagnetic resonance (EPR) spectra display anisotropic signals. Low-temperature solid-state X-band EPR data at 4 K reveal rhombic values g z = 1.980(4), g x = 2. 380(4), and g y = 2.225(4), as well as a forbidden signal at g = 4.24 for the Delta M S = 2 half field transition, in accord with 2 having two weakly interacting metal centers. Utilizing an S = 1 model, full spin Hamiltonian simulation of the low-temperature EPR spectrum on the solid sample was achieved by applying a nonzero zero-field-splitting parameter ( D = 0.001 cm (-1)), which is consistent with an S = 0 ground state with a very closely lying S = 1 state. Solid-state magnetization data also corroborate well with our solid-state EPR data and reveal weak antiferromagnetic behavior ( J = -1.52(5) cm (-1)) over a 2-300 K temperature range at a field of 1 Tesla. Evidence for 2 being a masked "(PNP)Ni" scaffold originates from its reaction with N 2CPh 2, which traps the Ni(I) monomer in the form of a T-shaped species, Ni(PNPNNCPh 2), a system that has been structurally characterized. The radical nature of complex 2, or its monomer component, is well manifested through the plethora of cooperative H-X-type bond cleavage reactions, providing the nickel(II) hydride (PNP)NiH and the corresponding rare functionalities -OH, -OCH 3, -PHPh, and -B(catechol) integrated into the (PNP)Ni moiety in equal molar amounts. In addition to splitting H 2, compound 2 can also engage in homolytic X-X bond cleavage reactions of PhXXPh to form (PNP)Ni(XPh) (X = S or Se).     

Enthalpies of mixing and intermolecular interactions in N,N-dimethylformamide-chloroform systems at temperatures ranging between 288 and 308 K

The thermal effects of N,N-dimethylformamide (DMF) and chloroform (ChF) dissolution in the ChF-DMF system are calorimetrically determined at 288, 298, and 308 K over the range of compositions. The partial molar enthalpies of the components are also calculated, along with the values for the enthalpies of mixing. It is found that mixing is a strongly exothermic process and depends only slightly on temperature. The enthalpy parameters of double and triple interactions between DMF molecules in ChF, and between ChF molecules in DMF, are determined using virial expansions.     

The crystal structures of the calcium-bound con-G and con-T[K7gamma] dimeric peptides demonstrate a metal-dependent helix-forming motif

Short peptides that have the ability to form stable alpha-helices in solution are rare, and a number of strategies have been used to produce them, including the use of metal chelation to stabilize folding of the backbone. However, no example exists of a structurally well-defined helix stabilized exclusively through metal ion chelation. Conantokins (con)-G and -T are short peptides that are potent antagonists of N-methyl-D-aspartate receptor channels. While con-G exhibits no helicity alone, it undergoes a structural transition to a helical conformation in the presence of a variety of multivalent cations, especially Mg2+ and Ca2+. This complexation also results in antiparallel dimerization of two peptide helices in the presence of Ca2+, but not Mg2+. A con-T variant, con-T[K7gamma], displays very similar behavior. We have solved the crystal structures of both Ca2+/con-G and Ca2+/con-T [K7gamma] at atomic resolution. These structures clearly show the nature of the metal-dependent dimerization and helix formation and surprisingly also show that the con-G dimer interface is completely different from the con-T[K7gamma] interface, even though the metal chelation is similar in the two peptides. This represents a new paradigm in helix stabilization completely independent of the hydrophobic effect, which we define as the "metallo-zipper."     

Specificity of immobilized porcine pepsin in H/D exchange compatible conditions

Statistical analysis of data from 39 proteins (13 766 amino acid residues) digested with immobilized porcine pepsin under conditions compatible with hydrogen/deuterium (H/D) exchange (<1 degrees C, <30 s) was performed to examine pepsin cleavage specificity. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe and Leu are favored residues each with a cleavage probability greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. Pro also cannot be at position P2 (cleavage probability <0.3%). Occupation of the P3 position by His, Lys, or Arg, or occupation of the P2' position by Pro, also leads to very little cleavage (cleavage probability <1.7%). The average cleavage probability over the entire data set was 13.6%, which is slightly lower than the value previously obtained by Powers et al. (14.8%). This is due, in part, to the larger protein sizes used in the current study. While the specificity of pepsin was similar to that previously observed, higher selectivity was observed in the present study due to less experimental variation in the conditions used to generate our database.     

The effects of gamma rays upon monohydrated and anhydrous asparagine: a DSC study in sealed pans

Non-irradiated and gamma irradiated monohydrated (l Asn·H₂O) and anhydrous (l Asn) asparagines, in solid state, were studied by means of DSC. The samples were irradiated at room temperature with gamma radiations using a ¹³⁷Cs source. The exposure doses ranged between 1 and 10 kGy. All samples were scanned in sealed pans, from room temperature to a temperature beyond the melting point. The DSC scans of l Asn·H₂O samples in sealed crucibles revealed the presence of two dehydration processes and one of decomposition and only decomposition in the case of l Asn. The influence of gamma irradiation consisted in decreasing the enthalpy of dehydration and of decomposition. A decomposition mechanism is proposed.     

喜树碱类抗肿瘤药物作用模式的柔性分子对接研究

研究采用柔性分子对接技术，将15个喜树碱类化合物对接到拓扑异构酶I (Topo I)-DNA切割复合物中，从原子水平和分子力场角度阐明了喜树碱类抗肿瘤药 物与DNA，Topo I的相互作用机制。研究发现，喜树碱分子插入Topp I-DNA复合物 的切割位点，并与Asn722，Asp533，Lys532和Lys720形成氢键作用网络。定量构效 关系研究进一步表明喜树碱分子可以与Topo I-DNA切割复合物形成电荷迁移作用。 该对接模型系统解释了喜树碱类化合物的构效关系、定点突变等诸多实验事实，为 下一步设计、合成新型高效的喜树碱类衍生物打下了坚实基础。     

Photochemical nucleophile mapping: identification of Tyr311 within the catalytic domain of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

Photochemical mapping of nucleophiles in close proximity to the active site Cys149 of rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was demonstrated based on the nucleophilic aromatic photosubstitution reaction using two regioisomers of alkoxy-fluoro-nitro-substituted benzenes. Two photophores were covalently attached to the active site SH group of GAPDH and the protein was subjected to photolysis then to the cyanogen bromide cleavage reaction. The advantage of this method is the capability to chase labeled products by monitoring absorption at 380 nm because of the chromogenic property of photophore. HPLC separation identified a large labeled peptide fragment that was further digested by V8 protease for Edman sequence analysis. From the recent X-ray crystallography of rabbit GAPDH, Tyr311, His176, Ser238 and Lys183 are closely located to catalytic Cys149. Among these nucleophiles, Tyr311 was preferentially labeled with 2-fluoro-4-nitrophenoxy photophore and no label was identified with the isomeric 4-fluoro-2-nitrophenoxy photophore. The result clearly reflects the distance between Cys149 and nucleophiles to distinguish the nearest Tyr311. As photophores show great reactivity even with water under neutral conditions, the distance between nucleophiles and photophores is important for photoinduced nucleophilic aromatic substitution. The method will provide a useful technique to survey nucleophiles within the catalytic domain.     

Synthesis of 3-iodoindoles by the Pd/Cu-catalyzed coupling of N,N-dialkyl-2-iodoanilines and terminal acetylenes, followed by electrophilic cyclization

[reaction: see text] 3-Iodoindoles have been prepared in excellent yields by coupling terminal acetylenes with N,N-dialkyl-o-iodoanilines in the presence of a Pd/Cu catalyst, followed by an electrophilic cyclization of the resulting N,N-dialkyl-o-(1-alkynyl)anilines using I2 in CH2Cl2. Aryl-, vinylic-, alkyl-, and silyl-substituted terminal acetylenes undergo this process to produce excellent yields of 3-iodoindoles. The reactivity of the carbon-nitrogen bond cleavage during cyclization follows the following order: Me > n-Bu, Me > Ph, and cyclohexyl > Me. Subsequent palladium-catalyzed Sonogashira, Suzuki, and Heck reactions of the resulting 3-iodoindoles proceed smoothly in good yields.     

Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme–Mg-8-oxo-dGMP complex

The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a K_D=52 nM, (ΔG°=−9.8 kcal/mol) which is 6.1 kcal/mol tighter than the binding of dGMP (ΔG°=−3.7 kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable ΔH_binding (−32 kcal/mol) despite an unfavorable −TΔS°_binding (+22 kcal/mol). The solution structure of the MutT–Mg²⁺-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in ¹H–¹⁵N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3 kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1 kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8=O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position −119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3–0.4 kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT–Mg²⁺-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1 kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a single hydrogen bond to its C6=O. The R78K+N119A double mutant weakened the binding of 8-oxo-dGMP (K_I^slope=3.1 mM) by 6.5±0.2 kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0±0.3 kcal/mol). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT–Mg²⁺-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4±0.5 Å between their side chain nitrogens.