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PubMed1997

Isothermal titration microcalorimetric studies for the binding of octenoyl-CoA to medium chain acyl-CoA dehydrogenase.

We investigated the binding of octenoyl-CoA to pig kidney medium chain acyl-CoA dehydrogenase (MCAD) by isothermal titration microcalorimetry under a variety of experimental conditions. At 25 degrees C in 50 mM phosphate buffer at pH 7.6 (ionic strength of 175 mM), the binding is characterized by the stoichiometry (n) of 0.89 mole of octenoyl-CoA/(mole of MCAD subunit), delta G = -8.75 kcal/mol, delta H = -10.3 kcal/mol, and delta S = -5.3 cal mol(-1) K(-1), suggesting that formation of MCAD-octenoyl-CoA is enthalpically driven. By employing buffers with various ionization enthalpies, we discerned that formation of the MCAD-octenoyl-CoA complex, at pH 7.6, accompanies abstraction (consumption) of 0.52 +/- 0.15 proton/(MCAD subunit) from the buffer media. We studied the effects of pH, ionic strength, and temperature on the thermodynamics of MCAD-octenoyl-CoA interaction. Whereas the ionic strength does not significantly influence the above interaction, the pH of the buffer media exhibits a pronounced effect. The pH dependence of the association constant of MCAD +octenoyl-CoA <==> MCAD-octenoyl-CoA yields a pKa for the free enzyme of 6.2. Among thermodynamic parameters, whereas delta G remains invariant as a function of temperature, delta H and deltaS(standard) both decrease with an increase in temperature. At temperatures of < 25 degrees C, delta G is dominated by favorable entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attain a value of zero at 23.8 degrees C, and then becomes unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of delta H yields the heat capacity change (delta Cp(0)) of -0.37 +/- 0.05 kcal mol(-1) K(-1), attesting to the fact that the binding of octenoyl-CoA to MCAD is primarily dominated by the hydrophobic forces. The thermodynamic data presented herein are rationalized in light of structural-functional relationships in MCAD catalysis.

Acyl Coenzyme A↗
PubMed1997

Titration calorimetric analysis of AcylCoA recognition by myristoylCoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoylCoA:protein N-myristoyltransferase (Nmt1p) is an essential enzyme that catalyzes the transfer of myristic acid (C14:0) from myristoylCoA to the N-terminus of cellular proteins with a variety of functions. Nmts from an assortment of species display remarkable in vivo specificity for this rare acyl chain. To better understand the mechanisms underlying this specificity, we have used isothermal titration calorimetry as well as kinetic measurements to study the interactions of Nmt1p with acylCoA analogs having variations in chain length and/or conformation, analogs with alterations in the thioester bond, and analogs with or without a 3'-phosphate in their CoA moiety. MyristoylCoA binds to Nmt1p with a Kd of 15 nM and a large exothermic deltaH (-25 kcal/mol). CoA derivatives of C12:0-C16:0 fatty acids bind to Nmt1p with similar affinity, but with much smaller deltaH and a correspondingly less negative TdeltaS than myristoylCoA. Replacing the thioester carbonyl group with a methylene or removing the 3'-phosphate of CoA is each sufficient to prevent the low enthalpy binding observed with myristoylCoA. The carbonyl and the 3'-phosphate have distinct and important roles in chain length recognition over the range C12-C16. Acyltransferase activity parallels binding enthalpy. The naturally occurring cis-5-tetradecenoylCoA and cis-5,8-tetradecadienoylCoA are used as alternative Nmt substrates in retinal photoreceptor cells, even though they do not exhibit in vitro kinetic or thermodynamic properties that are superior to those of myristoylCoA. The binding of an acylCoA is the first step in the enzyme's ordered reaction mechanism. Our findings suggest that within cells, limitation of Nmt substrate usage occurs through control of acylCoA availability. This indicates that full understanding of how protein acylation is controlled not only requires consideration of the acyltransferase and its peptide substrates but also consideration of the synthesis and/or presentation of its lipid substrates.

Acyl Coenzyme A↗
PubMed1998

Thermodynamics of membrane partitioning for a series of n-alcohols determined by titration calorimetry: role of hydrophobic effects.

Recent studies have shown that the traditional paradigm relying on hydrophobic effects is not adequate to describe membrane partitioning of amphiphilic solutes. To elucidate the thermodynamics and determine the role of the hydrophobic effect in the partitioning of small amphiphilic molecules into lipid bilayers, we have used titration calorimetry to directly measure the enthalpy, partition coefficients, and heat capacity change for the partitioning of a series of n-alcohols into lipid bilayers of several lipid compositions. The incremental thermodynamic quantities have been compared with model compound data for partitioning into bulk hydrocarbon solvents. We have found that there is a large negative heat capacity change upon partitioning, indicating a major contribution from the dehydration of nonpolar solute moieties; however, these hydrophobic effects also involve changes in lipid interactions with water in the interfacial region of the bilayer. In addition, we have found that the enthalpy effects are large, indicating that the partitioning process is accompanied by significant changes in the intralipid interactions within the bilayer. Cholesterol has a large effect on partitioning thermodynamics, making both the enthalpy and entropy contributions significantly more positive, resulting in a relatively small net decrease in the negative free energy of partitioning. These results demonstrate that while hydrophobic interactions play a major role in partitioning, the process is considerably more complex than the partitioning of model compounds between water and bulk hydrocarbons, with major contributions coming from changes in the structure and thermodynamic state of the bilayer, including the interfacial region. The results are discussed in terms of both the thermodynamics of partitioning and the role of lipid properties in membrane function. Our results support a paradigm for membrane structure and function in which the thermodynamic state, which is a function of lipid composition, temperature, and dissolved solutes, is a critical membrane property.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1998

Thermodynamic analysis of the heparin interaction with a basic cyclic peptide using isothermal titration calorimetry.

Brain natriuretic peptide (BNP) was examined as part of a continuing study of the interaction of proteins and peptides with the glycosaminoglycan heparin. BNP was tentatively identified as a heparin-binding protein on the basis of its cyclic structure and the high frequency of the basic amino acid residues, lysine and arginine. Thermodynamic analysis using isothermal titration calorimetry confirmed heparin binding to BNP with a micromolar Kd. Surprisingly, despite the high frequency (22%) of basic residues in BNP, only a small portion of the free energy of this interaction resulted from ionic contributions under physiologic conditions. The contribution of polar amino acids, representing 28% of BNP, was next examined in a variety of different buffers. These experiments demonstrated the transfer of five protons from buffer to BNP on heparin binding, suggesting that hydrogen bonding between the polar residues of BNP and heparin is a major factor contributing to the free energy of BNP binding to heparin. Hydrophobic forces apparently play only a small role in binding. Heparin contains few nonpolar functional groups, and a positive change in heat capacity (DeltaCp = 1 kcal/mol) demonstrates the loss of polar residues on BNP-heparin binding.

Amino Acid Sequence↗
PubMed1998

Crystal structures of the G protein Gi alpha 1 complexed with GDP and Mg2+: a crystallographic titration experiment.

The effect of Mg2+ binding on the conformation of the inactive GDP-bound complex of the heterotrimeric G protein alpha subunit Gi alpha 1 has been investigated by X-ray crystallography. Crystal structures of the Gi alpha 1.GDP complex were determined after titration with 5, 10, 100, and 200 mM Mg2+. Comparison of these structures with that of the Mg2+-free complex revealed Mg2+ bound at the same site as observed in the structure of the active, Gi alpha 1. GTP gamma S.Mg2+-bound complex of Gi alpha 1, with a similar coordination scheme except for the substitution of a water molecule for an oxygen ligand of the gamma-phosphate of Gi alpha 1.GTP gamma S. Mg2+. In contrast to the GDP.Mg2+ complex of Gt alpha and of other G proteins, switch I residues of Gi alpha 1 participate in Mg2+ binding and undergo conformational changes as a consequence of Mg2+ binding. Partial order is induced in switch II, which is disordered in the Mg2+-free complex, but no order is observed in the switch III region. This contrasts with the GDP.Mg2+ complex of Gt alpha in which both switch II and III switch are ordered. Mg2+ binding also induces binding of an SO42- molecule to the active site in a manner which may mimic a Gi alpha 1.GDP.PO42-.Mg2+ product complex. Implications of these findings are discussed.

Animals↗
PubMed1999

Thermodynamics of reversible and irreversible unfolding and domain interactions of glucoamylase from Aspergillus niger studied by differential scanning and isothermal titration calorimetry.

The stability of three forms of glucoamylase from Aspergillus niger has been investigated by differential scanning and isothermal titration calorimetry: Glucoamylase 1 (GA1), which consists of a catalytic domain and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region; glucoamylase 2 (GA2), which lacks SBD; and a proteolytically cleaved glucoamylase (GACD), which contains the catalytic domain and part of the linker region. The structures of the catalytic domain with part of the linker region and of SBD are known from crystallography and NMR, respectively, but the precise spatial arrangement of the two domains in GA1 is unknown. To investigate the stability of the three glucoamylase forms, we unfolded the enzymes thermally by differential scanning calorimetry (DSC). Aggregation occurs upon heating GA1 and GA2 at pH values between 2.5 and 5.0, whereas no aggregation is observed at higher pH (5.5-7.5). At all pH values, the catalytic domain of GA1 and GA2 unfolds irreversibly, while SBD unfolds reversibly in the pH range 5. 5-7.5 where aggregation does not occur. The unfolding of the catalytic domain of all glucoamylase forms seems to follow an irreversible one-step mechanism with no observable reversible intermediates on the experimental time scale. SBD of GA1 unfolds reversibly, and the ratio between the van't Hoff and calorimetric enthalpies is 1.4 +/- 0.1. Assignment of peaks of the DSC profile to the domains at pH 7.5 is achieved by using two different ligands: Acarbose, a very strong inhibitor that binds exclusively to the catalytic domain, and beta-cyclodextrin, a small starch analogue of which 2 molecules bind solely to the two binding sites present in SBD. Differences are seen in the unfolding processes of GA1 and GA2 since the former unfolds with one peak at all pH values, while the calorimetric trace of the latter can be resolved into more peaks depending on pH and the chemical composition of the buffers. In general, peaks corresponding to unfolding of GA2 are more complex than the peaks of GA1 and GACD. Some part of GA2 unfolds before the rest of the molecule which may correspond to the linker region or a particular early unfolding part of the catalytic domain. This leads to the conclusion that the structure of the GA2 molecule has a larger cooperative unfolding unit and is less stable than the structures of GA1 and GACD and that the C-terminal part of the linker region has a destabilizing effect on the catalytic domain.

Acarbose↗
PubMed1999

Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry.

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthritis and cancer. Among these enzymes, stromelysin-1 can also activate the proenzymes of other MMPs, making it an attractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze the binding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses a hydroxamic acid group (pK(a) congruent with 8.7) to bind the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent with 4.7). Binding affinity increased dramatically as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino acid residue of the enzyme also becomes protonated upon binding. Similar experiments carried out with the H224N mutant gave strong evidence that this residue is histidine 224. DeltaG, DeltaH, DeltaS, and DeltaC(p) were determined for the three inhibitors at pH 6.7, and DeltaC(p) was used to obtain estimates of the solvational, translational, and conformational components of the entropy term. The results suggest that: (1) a polar group at the P1 position can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' substituents of certain sizes may trigger an entropically unfavorable conformational change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities alone.

Binding Sites↗
PubMed2000

Protonation states and pH titration in the photocycle of photoactive yellow protein.

Photoactive yellow protein (PYP) undergoes a light-driven cycle of color and protonation states that is part of a mechanism of bacterial phototaxis. This article concerns functionally important protonation states of PYP and the interactions that stabilize them, and changes in the protonation state during the photocycle. In particular, the chromophore pK(a) is known to be shifted down so that the chromophore is negatively charged in the ground state (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a). The photocycle involves changes of one or both of these protonation states. Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I(2)) intermediate state. Calculations allowing multiple H-bonding arrangements around the chromophore also have been carried out. In addition, ground-state pK(a) values of the chromophore have been measured by UV-visible spectroscopy for the wild-type and the same three mutants. Because of the unusual protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calculations of electrostatic interactions in proteins. Good agreement between experiment and theory can be obtained for the ground state provided the protein interior is assumed to have a relatively low dielectric constant, but only partial agreement between theory and experiment is obtained for the bleached state. We also present a reinterpretation of previously published data on the pH-dependence of the recovery of the ground state from the bleached state. The new analysis implies a pK(a) value of 6.37 for Glu46 in the bleached state, which is consistent with other available experimental data, including data that only became available after this analysis. The new analysis suggests that signal transduction is modulated by the titration properties of the bleached state, which are in turn determined by electrostatic interactions. Overall, the results of this study provide a quantitative picture of the interactions responsible for the unusual protonation states of the chromophore and Glu46, and of protonation changes upon bleaching.

Amino Acid Substitution↗
PubMed2002

Formation quotients of aluminum sulfate complexes in NaCF3SO3 media at 10, 25, and 50 degrees C from potentiometric titrations using a mercury/mercurous sulfate electrode concentration cell.

Mercury/mercurous sulfate electrode concentration cells (with liquid junction) are employed in this study to determine the formation constants of aluminum sulfate complexes, with the principal advantage that the change in the free sulfate concentration is measured directly without the need to know the standard potential of the electrode. Potentiometric titrations were conducted at temperatures of 10, 25, and 50 degrees C and ionic strengths of approximately 0.3, 0.5, and 1.0 molal in aqueous solutions of the inert 1:1 electrolyte sodium trifluoromethanesulfonate (NaTr). Stoichiometric molal formation quotients Q1 and Q2, respectively, for the reactions Al3+(aq) + SO4(2-)(aq) AlSO4+(aq) and Al3+(aq) + 2SO4(2-)(aq) <= >Al(SO4)2-(aq) were determined. The values of log Q1 obtained from this work in NaTr media at ionic strengths of 0.3 and 1.0 mol x kg(-1) and 50 degrees C (1.72 +/- 0.08 and 1.35 +/- 0.06, respectively) are in excellent agreement with the values (1.71 +/- 0.2 and 1.32 +/- 0.1) determined in NaCl media from the recent potentiometric study conducted in the same laboratory using a hydrogen electrode concentration cell by Ridley et al. (Ridley, M. K.; Wesolowski, D. J.; Palmer, D. A.; Kettler, R. M. Geochim. Cosmochim. Acta 1999, 62, 459-472). The value of log Q2 (2.05 +/- 0.05) in 1.0 mol x kg(-1) from this work is smaller than the value reported by Ridley et al. (2.6 +/- 0.5) but within the combined experimental error. Empirical isothermal equations are presented to permit calculation of the equilibrium quotients as a function of ionic strength (0-1 mol x kg(-1)), giving log K1 and log K2 values at 25 degrees C and infinite dilution of 3.84 +/- 0.12 and 5.58 + 0.09, respectively. The value for log K1 obtained in this study at 25 degrees C is bracketed within experimental uncertainty by values reported by Kryzhanovskii et al. (Kryzhanovskii, M. M.; Volokhov, Y. A.; Pavlov, L. N.; Eremin, N. I.; Mironov, V. E Zh. Prikl. Khim. 1971, 44,476-479) and Nishide and Tsuchiya (Nishide, T.; Tsuchiya, R. Bull. Chem. Soc. Jpn. 1965, 38, 1398-1400), namely, 3.89 and 3.73, respectively. All other literature values for the first aluminum sulfate association constant are considerably lower than these results, which is also true for the second association constant, although there are few experimental data available for the latter. Empirical equations are also presented for calculating values of log Q1 and log Q2 from 0 to 1 molal ionic strength and from 10 to 125 degrees C, spanning the range of most environmental conditions at which these reactions are important.

Alum Compounds↗
PubMed2002

Automatic buffer capacity model building for advanced interpretation of titration curves.

An automatic buffer capacity model building algorithm was developed. The objective of this algorithm is to characterize multiple buffer systems from experimental titration curves. Starting from a simple mathematical model that incorporates the available prior knowledge on the buffer system, the model is sequentially extended by incorporating additional monoprotic buffer equations. To select the most appropriate model from the resulting set of automatically built models, a model structure selection technique was constructed from existing methods.

Algorithms↗
PubMed2004

Determination of strong ligand sites in sewage effluent-impacted waters by competitive ligand titration with silver.

A competitive ligand titration, employing Ag+, is used to determine the binding capacity of the small amounts of strong ligands (SL) in natural water samples. Strong ligands are defined here as high-affinity binding sites for group 11 and 12 metals such as Cu(I), Hg(II), and Ag(I). In addition, the conditional binding strength (log K') is determined for Ag- and SL. Diethyldithiocarbamate (DEDC) is the competitive ligand employed. The system is set at constant pH (8.1), ionic strength (0.1 M), and excess-fixed DEDC (10 microM) to determine SLs with log K' for Ag+ of >10. Silver was chosen as the titrant metal because it binds predominantly with S(-II) versus other ligands and reduced sulfur is thought to comprise the majority of SLs in natural waters. A two-phase system, water and 1,2-dichloroethane (DCE), is required due to the insolubility of Ag-DEDC in water. Added silver partitions into Ag+ and Ag-SL in the aqueous phase and into Ag-DEDC in the DCE phase. An automated system is used to add aliquots of silver and measure Ag-DEDC by UV absorbance in the DCE phase and [Ag+] by specific ion electrode in the aqueous phase. Excess addition of silver and a "Gran's" analysis gives the binding capacity of SL. The stability constant can also be determined for each addition of silver for an overall one-site SL assumption. Cysteine was used to test the method, and urban waters revealed SL capacities from about 50 to 150 nM and log K'(Ag) of 11-12. An independent analysis of chromium-reducible sulfide correlates well with the SL capacity.

Binding Sites↗
PubMed2000

Isothermal titration calorimetry measurements of Ni(II) and Cu(II) binding to His, GlyGlyHis, HisGlyHis, and bovine serum albumin: a critical evaluation.

The binding of Ni(II) and Cu(II) to histidine, to the tripeptides GlyGlyHis and HisGlyHis, and to the protein bovine serum albumin has been studied by isothermal titration calorimetry (ITC) to determine the experimental conditions and data analysis necessary to reproduce literature values for the binding constants and thermodynamic parameters. From analysis of the ITC data, we find that there are two major considerations for the use of this method to accurately quantify metal ion interaction with biological macromolecules. First, to determine true pH-independent binding constants, ITC data must be corrected for metal ion competition with protons by accounting for the experimental pH and pKa values of the metal-binding residues. Second, metal interaction with the buffer (stability and enthalpy of formation of metal-buffer complex(es)) must be included in the analysis of the ITC data to determine the binding constants and the change in enthalpy. While it may be possible to use a buffer that forms only weak, and therefore negligible, complexes with the metal, a buffer that has a strong and well-characterized interaction has the benefit of suppressing metal ion hydrolysis and precipitation, and of allowing the quantification of high-affinity metal-binding sites on biological macromolecules. This study has also quantified the contribution of the N-terminal imidazole of HisGlyHis to the stability of the Cu(II) and Ni(II) complexes of this protein sequence and has provided new insight about Cu(II) binding to albumin.

Calorimetry↗
PubMed2006

Cu(I) dinuclear complexes with tripodal ligands vs monodentate donors: triphenylphosphine, thiourea, and pyridine. A 1H NMR titration study.

Complexes [PPh3Cu(Tr(Mes,Me))] (1), [PPh3Cu(Tr(Me,o-Py))] (2), and [PPh3Cu(Br(Mes)pz(o-Py))] (3) (Tr(Mes,Me) = hydrotris[1,4-dihydro-3-methyl-4-mesityl-5-thioxo-1,2,4-triazolyl]borate; Tr(Me,o-Py) = hydrotris[1,4-dihydro-4-methyl-3-(2-pyridyl)-5-thioxo-1,2,4-triazolyl]borate; Br(Mes)pz(o-Py) = hydro[bis(thioxotriazolyl)-3-(2-pyridyl)pyrazolyl]borate; PPh3 = triphenylphosphine) were synthesized by the reaction of dinuclear complexes [Cu(Tr(Mes,Me))]2, [Cu(Tr(Me,o-Py))]2, [Cu(Br(Mes)pz(o-Py))]2, and PPh3. 1-3 were characterized by 1H, 13C, and 31P NMR spectroscopy and ESI-mass spectrometry. Crystal structure analyses were performed for 1 and 2. Both complexes crystallize in the triclinic P space group with the metal in a slightly distorted tetrahedral geometry (S3P coordination) bound by a kappa3-S3 ligand and a PPh3 molecule. The solution molecular structures were investigated by means of variable-temperature (210-310 K, CDCl3, 1-2; 200-310 K, CD2Cl2, 3) and NOESY NMR spectroscopy. The solution structures of 1-2 are in accordance with the X-ray structures, and the complexes do not exhibit fluxional behavior. On the other hand, 3 is subject to an equilibrium between two species with a coalescing temperature of approximately 260 K. DFT geometry optimizations suggest that the major species of 3 consists of the Br(Mes)pz(o-Py) ligand bound to Cu(I) in the kappa3-S2H fashion with two C=S groups and a [Cu...H-B] interaction. A PPh3 completes the copper coordination (S2HP coordination). The complex [TuCu(Tr(Mes,Me))] (4) (Tu = thiourea) was crystallized using an excess of Tu with respect to [Cu(Tr(Me,2-Py))]2 (approximately a 6:1 ratio). The metal adopts a distorted tetrahedral geometry with an overall S3H coordination determined by the bound kappa3-S2H ligand (two C=S groups and a [B-H...Cu] interaction) and by a Tu. The reactivity of dinuclear complexes [Cu(Tr(Mes,Me))]2, [Cu(Tr(Me,o-Py))]2, and [Cu(Br(Mes)pz(o-Py))]2 with monodentate ligands was investigated by means of NMR titrations with PPh3, Tu. and pyridine (Py), and formation constants for the adducts [DCu(L)] (D = monodentate donor, L = tripodal ligand) were determined.

Copper↗
PubMed2003

1H NMR titrations of hydroxy protons in aqueous solution as a method of investigation of intramolecular hydrogen-bonding in phosphorylated compounds: examples of myo-inositol 2-phosphate and myo-inositol 1,2,6-tris(phosphates).

1H NMR hydroxy proton titration experiments for myo-inositol 2-phosphate and myo-inositol 1,2,6-tris(phosphates) in aqueous solution are presented to demonstrate that by following OH signals versus pH, evidence can be brought for HB interaction between the hydroxyl and phosphate groups. The chemical shifts of the OH protons vicinal to phosphate groups appear deshielded by ca. 2.5 ppm with regard to those two centers removed from the phosphates. Remarkably, the deshielded protons are only present when their neighboring phosphate groups are fully deprotonated but persist until high pHs. From these results, C-OH...2-O3P-O type I, C-HO...-HO3P-O type II and C-OH...-HO3P-O type III hydrogen bonds are evidenced and discussed.

Hydrogen Bonding↗
PubMed2003

Quantification of protein-ligand interactions by mass spectrometry, titration, and H/D exchange: PLIMSTEX.

Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We report a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration, and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding constants obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concentration, the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concentrations of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and X-ray crystallography. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics.

Animals↗
PubMed2003

Titration in silico of reversible B <=> A transitions in DNA.

Reversible transitions between the A- and B-forms of DNA are obtained in free molecular dynamics simulations of a single double helix immersed in a water drop with Na(+) counterions. The dynamics of the transitions agrees with their supposed cooperative character. In silico titration of the transitions was carried out by smooth variation of the drop size. The estimated range of hydration numbers corresponding to the transition roughly agrees with experimental data. The chain length dependence was studied for double helices from 6 to 16 base pairs. It appeared that the B --> A transition is hindered for DNA shorter than one helical turn. With increased NaCl concentration in the drop, stabilization of the B-form is observed accompanied by the salt crystallization. The results strongly suggest that the B --> A transition at low hydration is caused by Na(+) ions sandwiched between phosphate strands in the major groove and is driven by direct medium range electrostatic interactions. The role of the reduced water shell apparently consists of increasing the counterion concentration in the opening of the major groove. Analysis of the available experimental data suggests that this mechanism is perhaps generally responsible for the A/B polymorphism in DNA.

Computer Simulation↗
PubMed2003

Understanding binding affinity: a combined isothermal titration calorimetry/molecular dynamics study of the binding of a series of hydrophobically modified benzamidinium chloride inhibitors to trypsin.

The binding of a series of p-alkylbenzamidinium chloride inhibitors to the serine proteinase trypsin over a range of temperatures has been studied using isothermal titration (micro)calorimetry and molecular dynamics simulation techniques. The inhibitors have small structural variations at the para position of the benzamidinium ion. They show small differences in relative binding affinity but large compensating differences in enthalpy and entropy. Binding affinity decreases with increased branching at the first carbon but increases with increasing the length of a linear alkyl substituent, suggesting that steric hindrance and hydrophobic interactions play dominant roles in binding. Structural analysis showed that the backbone of the enzyme was unaffected by the change of the para substituent. In addition, binding does not correlate strongly with octanol/water partition data. To further characterize this system, the change in the heat capacity on binding, the change in solvent-accessible surface area on binding, the effect of inhibitor binding on the hydration of the active site, the pK(a) of His57, and interactions within the catalytic triad have been investigated. Although the changes in inhibitor structure are small, it is demonstrated that simple concepts such as steric hindrance, hydrophobicity, and buried surface area are insufficient to explain the binding data. Other factors, such as access to the binding site and the cost of dehydration of the active site, are of equal or greater importance.

Benzamidines↗
PubMed2004

In situ FTIR-ATR analysis and titration of carboxylic acid-terminated SAMs.

FTIR-ATR measurements permit detailed structural analysis and in situ titration of carboxylate-terminated self-assembled monolayers. Both monomeric and dimeric/oligomeric acid groups are seen, and their acid-base behavior is directly monitored. Monomers that are hydrogen bonded only to surrounding water molecules have a pKa = 4.9, while the pKa for the aggregated molecules is 9.3.

Journal Article↗