Equilibrium studies on the binding of cadmium(II) to human serum transferrin

The binding of cadmium(II) to human serum transferrin in 0.01 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid with 5 mM bicarbonate at 25 degrees C has been evaluated by difference ultraviolet spectroscopy. Equilibrium constants were determined by competition versus three different low molecular weight chelating agents: nitrilotriacetic acid, ethylenediamine-N,N'-diacetic acid, and triethylenetetramine. Conditional equilibrium constants for the sequential binding of two cadmium ions to transferrin under the stated experimental conditions are log K1 = 5.95 +/- 0.10 and log K2 = 4.86 +/- 0.13. A linear free energy relationship for the complexation of cadmium and zinc has been prepared by using equilibrium data on 243 complexes of these metal ions with low molecular weight ligands. The transferrin binding constants for cadmium and zinc are in good agreement with this linear free energy relationship. This indicates that the larger size of the cadmium(II) ion does not significantly hinder its binding to the protein.     

Binding of retinoids and beta-carotene to beta-lactoglobulin. Influence of protein modifications

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.     

Different effects of lipid chain length on the two sides of a membrane and the lipid annulus of MscL

Quenching of the fluorescence of Trp residues in a membrane protein by lipids with bromine-containing fatty acyl chains provides a powerful technique for measuring lipid-protein binding constants. Single Trp residues have been placed on the periplasmic and cytoplasmic sides of the mechanosensitive channel of large conductance MscL from Mycobacterium tuberculosis to measure, separately, lipid binding constants on the two faces of MscL. The chain-length dependence of lipid binding was found to be different on the two sides of MscL, the chain-length dependence being more marked on the cytoplasmic than on the periplasmic side. To determine if lipid binding constants are affected by the properties of the lipid molecules not in direct contact with MscL (the bulk lipid), the amount of bulk lipid present in the system was varied. The binding constant of the short-chain phospholipid didodecylphosphatidylcholine was found to be independent of the molar ratio of lipid/MscL pentamer over the range 500:1-50:1, suggesting that lipid binding constants are determined largely by the properties of the lipid molecules interacting directly with MscL. These results point to a model in which lipid molecules located on the transmembrane surface of a membrane protein (the annular lipid molecules), by playing a dominant role in the interaction between a membrane protein and the surrounding lipid bilayer, could effectively buffer the membrane protein from changes in the properties of the bulk lipid bilayer.     

Estimation of binding constants by capillary electrophoresis

Capillary electrophoresis (CE) has become a useful technique for measuring binding constants. This review is focused on recent trends in the estimation of binding constants by affinity CE. First, we introduce several mathematical equations in which it is assumed that the stoichiometry of the binding between drug and protein is 1:1 as a simple model. In order to calculate accurate binding constants by affinity CE, several experimental considerations are described in this review. In addition, some recent methodologies, such as partial filling technique and multiple-step ligand injection method, are introduced. Among research publications within 3 years, recent applications for determining binding constants are reviewed.     

Kinetics and consequences of binding of nona- and dodecapeptides to the oligopeptide binding protein (OppA) of Lactococcus lactis.

The oligopeptide transport system (Opp) of Lactococcus lactis belongs to the class of binding protein-dependent ABC-transporters. This system has the unique capacity to mediate the uptake of peptides from 4 up to at least 18 residues. Kinetic analysis of peptide binding to the binding protein, OppA, revealed a relationship between the peptide dissociation constants and the length of the ligand. The dissociation constants varied from submicromolar for dodecapeptides to millimolar for pentapeptides. This implies that the residues 6-12 of the peptide contribute to the binding affinity, and, in contrast to the current views on peptide binding by homologous proteins, these residues must interact with OppA. Analysis of pre-steady-state kinetics of binding showed that the observed differences in the -values result primarily from variations in the dissociation rate constants. These results are discussed in relation to the affinity constant for transport of these substrates. Overall, the data suggest that the slow dissociation rate constants for the larger peptides are rate determining in the translocation of peptides across the membrane.     

Interactions of lipids and proteins: Some general principles

Methods to describe the binding of phospholipids to membrane proteins are described. It is shown that it is difficult to obtain estimates of the number of phospholipids bound to the surface of a membrane protein from ESR experiments in which plots of free to bound spin label (y) vs. molar ratio of lipid to protein are extrapolated toy=0. The relative advantages and disadvantages of ESR and fluorescence methods for measuring relative binding constants of phospholipids to membrane proteins are discussed. The particular problems associated with comparing binding constants of molecules of very different sizes (e.g., fatty acids and cardiolipin) are described and equations are presented to account for these problems. The possible effects of membrane viscosity and thickness on activity of membrane proteins are discussed, but it is concluded that effects of phospholipid structure on activity can only be understood in terms of a reasonably complete kinetic model for the protein.     

Equilibrium and kinetic aspects of protein-DNA recognition.

The specificity of regulatory protein binding to DNA is due to a complementarity between the sequence of reaction centres on the protein and the base pair sequence in the specific DNA site allowing the formation of a number of specific noncovalent bonds between the interacting entities. In the present communication the thermodynamic and kinetic aspects of these interactions are considered. The extent of binding specificity is shown to increase with an increase of the bond stability constants and with an increase in the number of ligand reaction centres. Kinetic analysis is carried out assuming that association process is very fast and that dissociation of nonspecific complexes is a rate-limiting step in the recognition of a specific binding site on DNA. The calculations show that a ligand can recognize its specific binding site on DNA within a reasonably limited time interval if the number of its reaction centres and the corresponding stability constants are strongly limited.     

Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein–DNA complexes and determining the stoichiometries and affinities of protein–protein complexes. We extend the capabilities of AFM to the determination of protein–DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein–DNA complexes.     

Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay

We have investigated the question of whether the gel mobility-shift assay can provide data that are useful to the demonstration of cooperativity in the site-specific binding of proteins to DNA. Three common patterns of protein-DNA interaction were considered: (i) the cooperative binding of a protein to two sites (illustrated by the Escherichia coli Gal repressor); (ii) the cooperative binding of a bidentate protein to two sites (illustrated by the E. coli Lac repressor); and (iii) the cooperative binding of a protein to three sites (illustrated by the lambda cI repressor). A simple, rigorous, and easily extendable statistical mechanical approach to the derivation of the binding equations for the different patterns is presented. Both simulated and experimental data for each case are analyzed. The mobility-shift assay provides estimates of the macroscopic binding constants for each step of ligation based on its separation of liganded species by the number of ligands bound. Resolution of the binding constants depends on the precision with which the equilibrium distribution of liganded species is determined over the entire range of titration of each of the sites. However, the evaluation of cooperativity from the macroscopic binding constants is meaningful only for data that are also accurate. Some criteria that are useful in evaluating accuracy are introduced and illustrated. Resolution of cooperative effects is robust only for the simplest case, in which there are two identical protein binding sites. In this case, cooperative effects of up to 1,000-fold are precisely determined. For heterogeneous sites, cooperative effects of greater than 1,000-fold are resolvable, but weak cooperativity is masked by the heterogeneity. For three-site systems, only averaged pair-wise cooperative effects are resolvable.     

An ultrafiltration assay for nucleotide binding to ribonucleotide reductase

Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.     

Binding kinetics of engineered mutants provide insight about the pathway for entering and exiting the intestinal fatty acid binding protein

To better understand the mechanism by which fatty acids bind to and dissociate from the binding cavities of fatty acid binding proteins (FABPs), we constructed 31 single amino acid mutants of the intestinal FABP (I-FABP) and determined the rate constants for binding and dissociation, primarily for long-chain fatty acids (FA). FA dissociation from these proteins was measured both by the ADIFAB method and by the change in tryptophan fluorescence of the FABPs. Rate constants for binding (kon) were calculated from the rate constants for dissociation (koff) and the equilibrium binding affinities. Amino acid substitutions were made at locations within the binding cavity, in the region of the gap between the betaD- and betaE-strands, and within the "portal" region of the protein. The koff values for the mutant proteins ranged from about 20-fold slower to 4-fold faster than the wild-type (WT) protein. Values for kon were as much as 20-fold slower than the WT protein, but in no case was kon significantly faster than the WT. Mutants with slower and faster koff values were generally those involving sites within the binding cavity and, relative to the WT protein, revealed higher and lower affinities, respectively. Reduced rates of binding were generally, but not exclusively, associated with sites within the portal region. For example, for F68A which is located closer to the opposite end of the protein from the portal region, the kon is more than 10-fold slower than WT. Even for these distal sites, however, the evidence is consistent with reductions in kon being due to alterations of the portal region. Binding affinities and rate constants measured as a function of ionic strength also suggest that the FA initially binds, through an electrostatic interaction, to Arg-56 on the surface of the protein, before inserting into the binding cavity. Thus, the results of this study are consistent with FA binding to I-FABP involving an initial interaction with Arg-56 followed by insertion of the FA, through the portal region, into the binding cavity and with a reversal of these steps for the dissociation reaction.     

Anatagonist potency and receptor binding.

Receptor binding constants, using 3H-dihydromorphine and P₂ fraction of rat brain homogenate, have been determined for 28 narcotic antagonists. A good correlation (R = 0.92) has been obtained between the binding constants and antagonist potency as determined by Kosterlitz et al. Using the guinea pig ileum preparation. It appears that the systems used in the correlation are useful in determining s similarities or dissimilarities of guinea pig ileum and rat brain receptors.     

Investigating protein unfolding kinetics by pulse proteolysis

Investigation of protein unfolding kinetics of proteins in crude samples may provide many exciting opportunities to study protein energetics under unconventional conditions. As an effort to develop a method with this capability, we employed “pulse proteolysis” to investigate protein unfolding kinetics. Pulse proteolysis has been shown to be an effective and facile method to determine global stability of proteins by exploiting the difference in proteolytic susceptibilities between folded and unfolded proteins. Electrophoretic separation after proteolysis allows monitoring protein unfolding without protein purification. We employed pulse proteolysis to determine unfolding kinetics of E. coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H). The unfolding kinetic constants determined by pulse proteolysis are in good agreement with those determined by circular dichroism. We then determined an unfolding kinetic constant of overexpressed MBP in a cell lysate. An accurate unfolding kinetic constant was successfully determined with the unpurified MBP. Also, we investigated the effect of ligand binding on unfolding kinetics of MBP using pulse proteolysis. On the basis of a kinetic model for unfolding of MBP•maltose complex, we have determined the dissociation equilibrium constant (K_d) of the complex from unfolding kinetic constants, which is also in good agreement with known K_d values of the complex. These results clearly demonstrate the feasibility and the accuracy of pulse proteolysis as a quantitative probe to investigate protein unfolding kinetics.     

Nonlinear Regression Analysis of the time Course of Ligand Binding Experiments

Abstract

The binding and release of hormones and growth factors are often relatively slow processes under biological conditions. Consequently, a knowledge of the underlying rate constants may be of greater physiological relevance than the equilibrium constant. Here we show how, by following a single time course of binding, the rate constants for both binding and release can be determined. The ratio of these rate constants allows the binding constant to be calculated. A nonlinear regression computer program is described which facilitates these calculations and which provides estimates and standard errors of the constants determined. The method is illustrated by the binding of human growth hormone to the human growth hormone binding protein, and the binding of ovine prolactin to the rabbit prolactin receptor.     

The 33 kDa protein of photosystem II is a low-affinity calcium- and lanthanide-binding protein

We have shown that the isolated 33 kDa protein of photosystem II contains one calcium and one lanthanide low-affinity binding site with binding constants (K(D)) on the order of 10(-5) M. Binding of calcium or lanthanides to this site induces conformational changes in the protein that manifest in fluorescence emission spectra of the protein, circular dichroism spectra, and calorimetric thermograms where the phase transitions are shifted to lower temperatures. The role of calcium binding to the 33 kDa protein in the attainment of its native structure and the significance of this interaction for the oxygen evolution process are discussed.     

Pt(CN)2-4 and Au(CN)-2: potential general probes for anion-binding sites of proteins. 35Cl and 81Br nuclear-magnetic-resonance studies.

Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35Cl- and 81Br- in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, alpha-chymotrypsin, human carbonic anhydrase, fructose-1,6-bisphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35Cl- and 81Br- relaxation rate on addition of functional ligands. Large, kinetically inert, complex anions like Pt(CN)2-4 and Au(CN)-2 are found to act as strong competitors towards halogen ions for the high-affinity anion binding sites of a number of proteins. Titrations with complex anions following the 35Cl- or 81Br- relaxation rates are found to be helpful in attempts to elucidate binding mechanisms. Especially, the complex anions may be useful probes for the discrimination between general and metallic anion binding sites in proteins and they also permit correlation of information from X-ray investigations of crystals with that from physical measurements in solution. From the change in halide ion quadrupole relaxation rate on addition of strongly binding ligands the quadrupole coupling constants of the high affinity Cl- and Br- binding sites are estimated using certain assumptions. It is found that for several proteins, comprising the metal-free proteins but also alcohol dehydrogenase and Escherichia coli alkaline phosphatase, the 35Cl quadrupole coupling constants have approximately the same values. For some other metallo-proteins like carbonic anhydrase and a zinc - serum-albumin complex considerably greater quadrupole coupling constants were obtained. The estimated quadrupole coupling constants are used as a basis for a discussion of the interactions involved in anion-protein interactions.     

Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli

The product of the rhaR gene, which regulates the level of mRNA produced from the four L-rhamnose-inducible promoters of the rhamnose operon, has been hypersynthesized and purified by a two-column procedure. The purified protein is a 33 kDa DNA-binding protein that binds to an inverted repeat structure located within the psr promoter, the promoter for the rhaS and rhaR genes. The equilibrium binding constants and kinetic constants have been determined under a variety of solution conditions. The protein binds with high affinity and its binding is sensitive to salt concentration and the presence of L-rhamnose. The nucleotides and phosphate residues contacted by RhaR were identified by chemical interference assays. All of the contacts are made to one face of the DNA and the symmetrical pattern matches the inverted repeat sequence proposed for the binding site. An unusual property of the binding site is that the two half-sites of the inverted repeat are separated from one another by 17 base-pairs of uncontacted DNA. Significant binding is retained if the 17 base-pairs are extended by insertions of integral turns of DNA, but not by half-integral turns. The complex of RhaR-DNA appears to be sharply bent, approximately 160 degrees.     

Kinetic analysis of site-specific photoaptamer-protein cross-linking

ssDNA oligonucleotides containing bromodeoxyuridine, BrdU-photoaptamers, are rapidly emerging as specific protein capture reagents in protein microarray technologies. A mathematical model for the kinetic analysis of photoaptamer-protein photocross-linking reactions is presented. The model is based on specific aptamer/protein binding followed by laser excitation that can lead to either covalent cross-linking of the photoaptamer and protein in the complex or irreversible photodamage to the aptamer. Two distinct kinetic regimes, (1) frozen and (2) rapid equilibrium, are developed analytically to model binding kinetics between laser pulses. The models are used to characterize the photocross-linking between three photoaptamers and their cognate protein targets; photoaptamers 0650 and 0615 cross-link human basic fibroblast growth factor and 0518 cross-links HIV MN envelope glycoprotein. Data for cross-linking reaction yields as a function of both laser energy dose and target protein concentration are analyzed for affinity constants and cross-link reaction rates. The binding dissociation constants derived from the cross-linking data are in good accord with independent measurements; the rapid equilibrium model appears to produce results more consistent with the experimental observations, although there is significant overlap between the two models for most conditions explored here. The rate of photodamage for 0615 and 0518 is 3.5 and 2.5 times that of the specific cross-link, giving low maximum reaction yields of approximately 20% and approximately 30%, whereas 0650 cross-links with a rate over five times higher than its photodamage rate and has a maximum reaction yield exceeding 80%. Quantum yields for the three systems are estimated from the data; photoaptamer 0650 has a reasonably high quantum yield of approximately 0.2 for protein cross-linking, while 0518 and 0615 have quantum yields of 0.07 and 0.02. The work presented here provides a useful set of metrics that allow for refinement of photoaptamer properties.     

Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains

Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.