Selectivity and specificity of substrate binding in methionyl-tRNA synthetase

The accuracy of in vivo incorporation of amino acids during protein biosynthesis is controlled to a significant extent by aminoacyl-tRNA synthetases (aaRS). This paper describes the application of the HierDock computational method to study the molecular basis of amino acid binding to the Escherichia coli methionyl tRNA synthetase (MetRS). Starting with the protein structure from the MetRS cocrystal, the HierDock calculations predict the binding site of methionine in MetRS to a root mean square deviation in coordinates (CRMS) of 0.55 A for all the atoms, compared with the crystal structure. The MetRS conformation in the cocrystal structure shows good discrimination between cognate and the 19 noncognate amino acids. In addition, the calculated binding energies of a set of five methionine analogs show a good correlation (R(2) = 0.86) to the relative free energies of binding derived from the measured in vitro kinetic parameters, K(m) and k(cat). Starting with the crystal structure of MetRS without the methionine (apo-MetRS), the putative binding site of methionine was predicted. We demonstrate that even the apo-MetRS structure shows a preference for binding methionine compared with the 19 other natural amino acids. On comparing the calculated binding energies of the 20 natural amino acids for apo-MetRS with those for the cocrystal structure, we observe that the discrimination against the noncognate substrate increases dramatically in the second step of the physical binding process associated with the conformation change in the protein.     

Aminoacylation of tRNAs as critical step of protein biosynthesis.

Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases. The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles. Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps. Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000. Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme. A simple hypothetical model of the isoleucine binding site is postulated. Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology. Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA. Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction. The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted.     

Computational analysis of binding of P1 variants to trypsin

The binding of P1 variants of bovine pancreatic trypsin inhibitor (BPTI) to trypsin has been investigated by means of molecular dynamics simulations. The specific interaction formed between the amino acid at the primary binding (P1) position of the binding loop of BPTI and the specificity pocket of trypsin was estimated by use of the linear interaction energy (LIE) method. Calculations for 13 of the naturally occurring amino acids at the P1 position were carried out, and the results obtained were found to correlate well with the experimental binding free energies. The LIE calculations rank the majority of the 13 variants correctly according to the experimental association energies and the mean error between calculated and experimental binding free energies is only 0.38 kcal/mole, excluding the Glu and Asp variants, which are associated with some uncertainties regarding protonation and the possible presence of counter-ions. The three-dimensional structures of the complex with three of the P1 variants (Asn, Tyr, and Ser) included in this study have not at present been solved by any experimental techniques and, therefore, were modeled on the basis of experimental data from P1 variants of similar size. Average structures were calculated from the MD simulations, from which specific interactions explaining the broad variation in association energies were identified. The present study also shows that explicit treatment of the complex water-mediated hydrogen bonding network at the protein-protein interface is of crucial importance for obtaining reliable binding free energies. The successful reproduction of relative binding energies shows that this type of methodology can be very useful as an aid in rational design and redesign of biologically active macromolecules.     

Stability of monomeric Cro variants: Isoenergetic transformation of a type I' to a type II' beta-hairpin by single amino acid replacements

The thermodynamic stabilities of three monomeric variants of the bacteriophage lambda Cro repressor that differ only in the sequence of two amino acids at the apex of an engineered beta-hairpin have been determined. The sequences of the turns are EVK-XX-EVK, where the two central residues are DG, GG, and GT, respectively. Standard-state unfolding free energies, determined from circular dichroism measurements as a function of urea concentration, range from 2.4 to 2.7 kcal/mole, while those determined from guanidine hydrochloride range from 2.8 to 3.3 kcal/mole for the three proteins. Thermal denaturation yields van't Hoff unfolding enthalpies of 36 to 40 kcal /mole at midpoint temperatures in the range of 53 to 58 degrees C. Extrapolation of the thermal denaturation free energies with heat capacities of 400 to 600 cal/mole deg gives good agreement with the parameters determined in denaturant titrations. As predicted from statistical surveys of amino acid replacements in beta-hairpins, energetic barriers to transformation from a type I' turn (DG) to a type II' turn (GT) can be quite small.     

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

By combining crystallographic and NMR structural data for RNA-bound amino acids within riboswitches, aptamers, and RNPs, chemical principles governing specific RNA interaction with amino acids can be deduced. Such principles, which we summarize in a “polar profile”, are useful in explaining newly selected specific RNA binding sites for free amino acids bearing varied side chains charged, neutral polar, aliphatic, and aromatic. Such amino acid sites can be queried for parallels to the genetic code. Using recent sequences for 337 independent binding sites directed to 8 amino acids and containing 18,551 nucleotides in all, we show a highly robust connection between amino acids and cognate coding triplets within their RNA binding sites. The apparent probability (P) that cognate triplets around these sites are unrelated to binding sites is ≅5.3 × 10⁻⁴⁵ for codons overall, and P ≅ 2.1 × 10⁻⁴⁶ for cognate anticodons. Therefore, some triplets are unequivocally localized near their present amino acids. Accordingly, there was likely a stereochemical era during evolution of the genetic code, relying on chemical interactions between amino acids and the tertiary structures of RNA binding sites. Use of cognate coding triplets in RNA binding sites is nevertheless sparse, with only 21% of possible triplets appearing. Reasoning from such broad recurrent trends in our results, a majority (approximately 75%) of modern amino acids entered the code in this stereochemical era; nevertheless, a minority (approximately 21%) of modern codons and anticodons were assigned via RNA binding sites. A Direct RNA Template scheme embodying a credible early history for coded peptide synthesis is readily constructed based on these observations.     

Amino acid conjugated sophorolipids: A new family of biologically active functionalized glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).     

Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding.

In a recent study, an RNA aptamer for the specific recognition of the amino acid L-arginine was evolved from an in vitro selected L-citrulline binding parent sequence [M. Famulok (1994) J. Am. Chem. Soc. 116, 1698-1706]. We have now carried out a structural analysis of these aptamers by using chemical modification experiments. Footprinting experiments and a damage selection approach were performed to identify those positions protected from modification in the presence of the amino acids and modifications that interfere with the binding of the ligand. It is shown that of the two bulged regions present in both aptamers one can be modified without loss of binding activity whereas in the other bulge nearly every position is shown to be involved in the recognition of the ligands. This might be indicative for non-canonical base pairing to occur within the non-Watson-Crick paired regions which might be stabilized by the complexed amino acid. Binding to the cognate amino acid significantly enhances the conformational stability of the RNA. We also tested the sensitivity of both aptamers towards lead (II) ion induced cleavage and identified a hypersensitive cleavage site within the invariant bulged region. Lead cleavage is inhibited by the complexed amino acid, indicating a conformational change of the aptamer upon ligand binding. NMR titration data obtained with both aptamers and their cognate ligands confirm the proposed conformational changes and indicate the formation of a 1:1 complex of RNA:amino acid.     

Calculations of free-energy contributions to protein-RNA complex stabilization

The problem of calculating binding affinities of protein-RNA complexes is addressed by analyzing a computational strategy of modeling electrostatic free energies based on a nonlinear Poisson-Boltzmann (NLPB) model and linear response approximation (LRA). The underlying idea is to treat binding as a two-step process. Solutions to the NLPB equation calculate free energies arising from electronic polarizability and the LRA is constructed from molecular dynamics simulations to model reorganization free energies due to conformational transitions. By implementing a consistency condition of requiring the NLPB model to reproduce the solute-solvent free-energy transitions determined by the LRA, a "macromolecule dielectric constant" (epsilon(m)) for treating reorganization is obtained. The applicability of this hybrid approach was evaluated by calculating the absolute free energy of binding and free-energy changes for amino acid substitutions in the complex between the U1A spliceosomal protein and its cognate RNA hairpin. Depending on the residue substitution, epsilon(m) varied from 3 to 18, and reflected dipolar reorientation not included in the polarization modeled by epsilon(m) = 2. Although the changes in binding affinities from substitutions modeled strictly at the implicit level by the NLPB equation with epsilon(m) = 4 reproduced the experimental values with good overall agreement, substitutions problematic to this simple treatment showed significant improvement when solved by the NLPB-LRA approach.     

Radiation disrupts protein-DNA complexes through damage to the protein. The lac repressor-operator system.

Eon, S., Culard, F., Sy, D., Charlier, M. and Spotheim-Maurizot, M. Radiation Disrupts Protein-DNA Complexes through Damage to the Protein. The lac Repressor-Operator System. Radiat. Res. 156, 110-117 (2001).Binding of a protein to its cognate DNA sequence is a key step in the regulation of gene expression. If radiation damage interferes with protein-DNA recognition, the entire regulation process may be perturbed. We have studied the effect of gamma rays on a model regulatory system, the E. coli lactose repressor-operator complex. We have observed the disruption of the complex upon irradiation in aerated solution. The complex is completely restored by the addition of nonirradiated repressor, but not by the addition of nonirradiated DNA. Thus radiation disrupts the DNA-protein complex by affecting the binding ability of the protein. This interpretation is supported by the dramatic loss of binding ability of a free irradiated repressor toward nonirradiated DNA. Interestingly, the dose necessary for the disruption of the irradiated complex is higher than that for inducing the complete loss of the binding ability of the free irradiated repressor. This may be due to the protection of key amino acids by the bound DNA. As seen from calculations of the accessibility of amino acids to radiolytic OH(.), the protection is due to both masking and conformational effects.     

Developmental changes of glucocorticoid receptors in the rat pancreas

Glucocorticoids are known to play a role in the maturation of the exocrine pancreas. The exact mechanism of glucocorticoid action in pancreatic ontogeny is, however, not clear. The present study characterized and quantitated the binding of [3H]dexamethasone to cytosol fractions from pancreata of rats at various ages. Trunk blood samples from these rats were also checked for levels of free and bound corticosterone. Specific and saturable bindings for dexamethasone were found in pancreatic cytosol fractions from newborn suckling and adult rats. Competition studies showed a preference for steroids with glucocorticoid activity. Specific binding was relatively low in pancreatic cytosol from newly born and 1-day old pups. A significant rise was seen after day 15. Cytosolic binding capacities were greatest from pancreata obtained from pups at weaning (3rd to 5th weeks). Values then declined toward the adult level. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 7.3 (+/- 1.1) X 10(-8) M and number of binding sites equalled to 1.29 (+/- 0.18) X 10(-13) mole/mg of cytosolic protein in adult rat pancreas. Pancreata from 25- and 15-day old rats had Kds of 3.4 (+/- 0.8) X 10(-8) M and 2.7 (+/- 0.7) X 10(-8) M with the number of binding sites equal to 1.77 (+/- 0.21) X 10(-13) mole/mg protein and 1.31 (+/- 0.16) X 10(-13) mole/mg protein respectively. Total plasma corticosterone concentration was low before day 10. It rose significantly by day 15, peaked at day 25, and then declined after weaning. About 5-15% of corticosterone during weaning and about 20-30% before and after weaning were in the free form. The peak level of dexamethasone binding corresponded to an increase in the plasma corticosterone level during weaning. This suggests a close relationship between plasma corticosterone levels and pancreatic glucocorticoid receptors. Both may, therefore, play a role in pancreatic development in the rat.     

Biochemical properties of phosphatidylcholine-binding proteins that share common antigenic determinants with Fc gamma 2b receptor

Biochemical and immunological properties of biosynthetically radiolabeled phosphatidylcholine-(PC-) binding proteins were investigated. The PC-binding proteins were extracted from the detergent lysate of biosynthetically radiolabeled P388D1 cells by affinity chromatography on PC-Sepharose and filtered through a Sephadex G-100 gel column in the presence of 6 M urea. Isoelectric focusing of the gel-filtered materials in the presence of 6 M urea revealed the presence of a major protein component of pIe of 5.8 and minor heterogeneous cellular proteins. The yield of the electrofocused PC-binding proteins based on protein determination by Lowry's method ranged from 0.7 to 4 mg per 10(9) cells. The purified PC-binding proteins appeared to be tightly associated with Triton X-100 and phospholipids in the weight ratio of 0.57 and 0.05 g/g of proteins, respectively. The majority of lipids that could be extracted from the PC-binding proteins by chloroform/methanol (2:1 v/v) are free fatty acids, whereas lipids extracted from Pronase-treated PC-binding proteins contained phosphatidylethanolamine. By amino acid analysis, the purified PC-binding proteins were found to consist of a minimum of 417 amino acid residues, suggesting a minimum molecular weight of about 38 000 for this protein. Results of radiolabeling experiments with [3H]glucosamine and amino acid analysis both showed the presence of a mole of glucosamine per a mole of the PC-binding proteins, suggesting their glycoprotein nature. About 40% of the purified PC-binding proteins coprecipitated with monoclonal anti-Fc gamma 2bR antibody (2.4G2) in detergent-containing buffer, whereas only 6% of the isolated IgG binding proteins reacted with this antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.     

Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

A network of conserved intramolecular contacts defines the off-state of the transmembrane switch mechanism in a seven-transmembrane receptor

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G-protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M(1) muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high-affinity Zn(2+) binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism.     

Interaction of water vapour with twenty different poly-L-amino acids and their excess hydration in presence of sodium chloride

Using the isopiestic vapour pressure technique, the magnitudes of excess binding of water and NaCl per mole of twenty different poly-L-amino acid residues, respectively in the presence of different bulk molefractions (X2) of NaCl have been evaluated from the mathematical expressions for the Gibbs surface excesses. At certain high ranges of NaCl concentration, the plot of -Gamma1 (2) versus X1/X2 becomes linear, so that moles of water and NaCl, respectively bound per mole of amino acid residue can be evaluated. -Gamma(2)1 is the excess moles of H20 per mole of amino acid residue and X1 and X2 stand for mole fractions of the water and NaCl, respectively in the sample system. Also, using the integrated form of the Gibbs absorption equation, the values of standard free energy change (deltaG(0)) for the excess adsorption of NaCl per kg of poly-L-amino acids have been evaluated. These values are all positive as a result of positive excess hydration of polyamino acids. The standard free energy of excess hydration deltaG(0)hy (equal to -deltaG(0)) is negative due to spontaneous excess hydration of polyamino acid in the presence of a salt.     

Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae

Yoch, D. C. (South Dakota State University, Brookings), and R. M. Pengra. Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92:618-622. 1966.-The effect of exogenous amino acids and the free amino acid pool on the synthesis of the nitrogenase system of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes M5al) was investigated. When an actively N(2)-fixing culture was used to inoculate a medium containing a limiting concentration of NH(4) (+), an induction lag period was observed. When either a single amino acid or a mixture of amino acids was substituted at the same nitrogen concentration, growth was uninterrupted by the induction period. It appears that a step or steps in the formation of the nitrogenase system are repressed by NH(4) (+) and are not affected by amino acid N. The amino acids, far from repressing formation of nitrogenase as does NH(4) (+), actually stimulate its formation. It appears that both free and amino nitrogen are used simultaneously. The amino acids that served concomitantly with N(2) as a source of nitrogen were: aspartic acid, serine, threonine, leucine, and histidine. Of these amino acids, it was shown that aspartic acid is readily taken up by the cells. Of the amino acids not serving as an immediate nitrogen source, isoleucine is not taken up by the cells. The free amino acid pool of the cells was measured at the onset and termination of the induction period. Ninhydrin-positive material in the amino acid pool was depleted by 35% during the induction period.     

Recognition of a flipped base in a hairpinloop DNA by a small peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258-259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 x 10(6) variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use pi-pi stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 x 10(6) M(-1) at 25 degrees C and the resulting binding free energy change at 25 degrees C (DeltaG degrees (25)) was -8.2 kcal mol(-1). The binding of the peptide to AP2 was also analyzed and the resulting binding constant and DeltaG degrees (25) were about 4.2 x 10(4) M(-1) and -6.3 kcal mol(-1), respectively. The difference in the binding free energy changes (DeltaDeltaG degrees (25)) of 1.9 kcal mol(-1) was comparable to the values reported in other systems and was considered a consequence of the loss of pi-pi stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

肾脏疾病患者体液氨基酸模式

使用 DNS— cl( Dimethylamino-naphthylane-5-sulfony chloride)荧光试剂 ,标记氨基酸成 DNS— AAs( DNS_ Amino Acids) ,然后采用聚酰胺薄膜层析 ,制成具有 2 0余种 DNS— AAs的荧光薄膜层析图谱 ,选择其中所需要检测的氨基酸 ,分别经洗脱后在日本岛津 RF— 51 0荧光分光光度计上 ,进行定量检测 ,分析了 3 6例慢性肾炎患者 ,1 8例尿毒症患者血浆中 1 2种游离氨基酸和 3 5例慢性肾炎患者尿液中 1 3种游离氨基酸 ,分别与 2 9例正常人血浆和3 0例正常人尿液中游离氨基酸进行了比较 ,以此探讨肾脏疾病体液氨基酸模式的同时提供氨基酸治疗肾脏疾病的科学依据。     

Evaluation of protein-protein association energies by free energy perturbation calculations

The association energy upon binding of different amino acids in the specificity pocket of trypsin was evaluated by free energy perturbation calculations on complexes between bovine trypsin (BT) and bovine pancreatic trypsin inhibitor (BPTI). Three simulations of mutations of the primary binding residue (P(1)) were performed (P(1)-Ala to Gly, P(1)-Met to Gly and P(1)-Met to Ala) and the resulting differences in association energy (DeltaDeltaG(a)) are 2. 28, 5.08 and 2.93 kcal/mol for P(1)-Ala to Gly, P(1)-Met to Gly and to Ala with experimental values of 1.71, 4.62 and 2.91 kcal/mol, respectively. The calculated binding free energy differences are hence in excellent agreement with the experimental binding free energies. The binding free energies, however, were shown to be highly dependent on water molecules at the protein-protein interface and could only be quantitatively estimated if the correct number of such water molecules was included. Furthermore, the cavities that were formed when a large amino acid side-chain is perturbed to a smaller one seem to create instabilities in the systems and had to be refilled with water molecules in order to obtain reliable results. In addition, if the protein atoms that were perturbed away were not replaced by water molecules, the simulations dramatically overestimated the initial state of the free energy perturbations.