The measurement of negative charge content in cartilage using a colloid titration technique.

A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 mu eq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.     

Thermometric titration studies of the interaction of block polypeptides with surfactants

The interactions between surfactants and block polypeptides were investigated by titration calorimetry and CD. The polypeptides exhibited signs of an interaction only with surfactants bearing a charge opposite to the charge on the polymer. The stoichiometry of the resulting complex was determined to be approximately equal to the polymer net charge. In general, a decrease in helical content accompanied the interaction between the block polypeptides and the surfactants. Both positive and negative enthalpy changes were noted, depending on the heat of micelle formation. None of the thermal effects noted were preceded by polymer unfolding, as is characteristic of the interaction between surfactants and typical globular proteins.     

Electrostatic contributions to residue-specific protonation equilibria and proton binding capacitance for a small protein

Charge-charge interactions in proteins are important in a host of biological processes. Here we use 13C NMR chemical shift data for individual aspartate and glutamate side chain carboxylate groups to accurately detect site-specific protonation equilibria in a variant of the B1 domain of protein G (PGB1-QDD). Carbon chemical shifts are dominated by changes in the electron distribution within the side chain and therefore excellent reporters of the charge state of individual groups, and the data are of high precision. We demonstrate that it is possible to detect local charge interactions within this small protein domain that stretch and skew the chemical shift titration curves away from "ideal" behavior and introduce a framework for the analysis of such convoluted data to study local charge-charge interactions and electrostatic coupling. It is found that, due to changes in electrostatic potential, the proton binding affinity, Ka, of each carboxyl group changes throughout the titration process and results in a linearly pH dependent pKa value. This result could be readily explained by calculations of direct charge-charge interactions based on Coulomb's law. In addition, the slope of pKa versus pH was dependent on screening by salt, and this dependence allowed the selective study of charge-charge interactions. For PGB1-QDD, it was established that mainly differences in self-energy, and not direct charge-charge interactions, are responsible for shifted pKa values within the protein environment.     

Effects of humic substances on the oxidation of pentachlorophenol by peroxosulfate catalyzed by iron(III)-phthalocyanine-tetrasulfonic acid

In an attempt to enhance the oxidation of pentachlorophenol (PCP) in the Fe(III)-PcTS/KHSO5 system, the presence of added humic substances (HSs) was studied, investigating the chemical properties of HSs related to the enhancement in PCP oxidation by correlations with the degree of enhancement in PCP oxidation (%delta(PCP)60). The %delta(PCP)60 value increased with a decrease in the content of oxygen-containing functional groups, such as carboxylic acids. This indicated that HSs with a lower content of oxygen-containing functional groups would be useful for enhancing the oxidation of PCP. A negative correlation between %delta(PCP)60 and the kinetic constants of Fe(III)-PcTS self-oxidation indicated that the enhancement by added HSs could be attributed to the suppression of Fe(III)-PcTS deactivation by self-oxidation. Such a stabilization of Fe(III)-PcTS could be attributed to hydrophobic interactions between the catalyst and HSs.     

Determination of surface charge of some bacteria by colloid titration

The surface charge of bacteria is always negative except below pH 2. The negative charge depends mainly upon phosphate and carboxyl groups. The charge of six kinds of bacteria was determined by colloid titration between pH 2 and 11. When there is amino group on the cell surface, it is positive as ammonium cation between pH 2 and 10, and it combines with the negative groups to neutralize the negative charge. In the presence of formalin, the colloid titration results show that the free amine is blocked by formalin, and the amino group can be estimated by the difference between the two titration results. Above pH 10, the titration results of Escherichia coli and Salmonella typhi in the presence of formalin implied the existence of guanidyl group. The titration results of living bacterial suspensions were quite the same as those of heat-killed ones.     

Electrostatic interactions of fluorescein dyes with proteins

The interactions of a number of halogen derivatives of fluorescein with human carbonylhaemoglobin and human serum albumin have been studied. The binding affinities of these proteins were compared with the charge properties of the dyes. The charge properties, determined from titration curves. Hückel molecular orbital (HMO) calculations and the equilibria established between polar and non-polar phase testify to an important role of dipole moments of the halogen derivatives in their interactions with proteins.     

Co-evolving motions at protein-protein interfaces of two-component signaling systems identified by covariance analysis

Short-lived protein interactions determine signal transduction specificity among genetically amplified, structurally identical two-component signaling systems. Interacting protein pairs evolve recognition precision by varying residues at specific positions in the interaction surface consistent with constraints of charge, size, and chemical properties. Such positions can be detected by covariance analyses of two-component protein databases. Here, covariance is shown to identify a cluster of co-evolving dynamic residues in two-component proteins. NMR dynamics and structural studies of both wild-type and mutant proteins in this cluster suggest that motions serve to precisely arrange the site of phosphoryl transfer within the complex.     

On the temperature dependence of complex formation between chitosan and proteins

Chitosan is a biocompatible easily degradable polysaccharide, which, because of its positive charge, is able to interact favorably with deprotonated carboxyl groups of proteins. The strength of these charge-charge interactions is generally low, resulting in poor colloidal stability of the complexes. To investigate if other noncovalent forces contribute to stabilizing such systems, we have selected α-lactalbumin, β-lactoglobulin, β-casein, and human growth hormone, characterized by a common acidic pI value (～ 5) that ensures their overall negative charge at physiological pH. Binding energetics between chitosan and proteins was studied by isothermal titration calorimetry, whereas the thermal stability was assessed by differential scanning calorimetry. Our data show that colloidal stability of the particles depends on protein identity as well as temperature, indicating the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) as driving forces for the complex formation. This suggests that chitosan-protein drug delivery systems can be improved through preparation process optimization with regard to temperature.     

The polyelectrolyte properties of elastin.

The charge structure and ionic interactions of elastin prepared from the pig thoracic aorta by acid, alkali, or CNBr extraction have been investigated by potentiometric titration and radiotracer techniques. The number of charged groups was consistent with the amino acid composition, comparable to elastin from other sources and insensitive to the method of preparation. The enthalpies of ionization of the basic groups were comparable for those previously found for proteins but those of the acidic groups were higher. Ionic interactions were predominantly electrostatic although a strong affinity for chloride ions was noted. Changes in ionic interactions as the elastin was stretched had a similar effect to an increase in the apparent fixed charge density of the tissue. Mechanical strain altered the protonation of the elastin and the pK of the carboxyl groups. Conversely, the conformation of the elastin network varied with ionic strength and pH, being particularly sensitive to the degree of ionization of the more basic groups and with the ionic strength and anion composition of the medium. We speculate that strain induced changes in the conformation of elastin altering its reactivity towards lipids, ions or matrix macromolecules or changes in its mechanical properties resulting from changes in its ionic environment may be of physiological or pathological importance.     

Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge −19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT_trs/d[ion] (T_trs; thermal midpoint). We show that dT_trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.     

A Missense Mutation in the Aggrecan C-type Lectin Domain Disrupts Extracellular Matrix Interactions and Causes Dominant Familial Osteochondritis Dissecans

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.     

Probing the energetic and structural role of amino acid/nucleobase cation-pi interactions in protein-ligand complexes

X-ray structures of proteins bound to ligand molecules containing a nucleic acid base were systematically searched for cation-pi interactions between the base and a positively charged or partially charged side chain group located above it, using geometric criteria. Such interactions were found in 38% of the complexes and are thus even more frequent than pi-pi stacking interactions. They are moreover well conserved in families of related proteins. The overwhelming majority of cation-pi contacts involve Ade bases, as these constitute by far the most frequent ligand building block; Arg-Ade is the most frequent cation-pi pair. Ab initio energy calculations at MP2 level were performed on all recorded pairs. Though cation-pi interactions involving the net positive charge carried by Arg or Lys side chains are the most favorable energetically, those involving the partial positive charge of Asn and Gln side chain amino groups (sometimes referred to as amino-pi interactions) are favorable too, owing to the electron correlation energy contribution. Chains of cation-pi interactions with a nucleobase bound simultaneously to two charged groups or a charged group sandwiched between two aromatic moieties are found in several complexes. The systematic association of these motifs with specific ligand molecules in unrelated protein sequences raises the question of their role in protein-ligand structure, stability, and recognition.     

Osmotic loading to determine the intrinsic material properties of guinea pig knee cartilage

Few methods exist to study cartilage mechanics in small animal joints due to the difficulties associated with handling small tissue samples. In this study, we apply an osmotic loading method to quantify the intrinsic material properties of articular cartilage in small animal joints. Cartilage samples were studied from the femoral condyle and tibial plateau of two-month old guinea pigs. Swelling strains were measured using confocal fluorescence scanning microscopy in samples subjected to osmotic loading. A histochemical staining method was developed and calibrated for quantification of negative fixed charge density in guinea pig cartilage. Site-matched swelling strain data and fixed charge density values were then used with a triphasic theoretical model for cartilage swelling to determine the uniaxial modulus of the cartilage solid matrix. Moduli obtained in this study (7.2 MPa femoral condyle; 10.8 MPa, tibial plateau) compare well with previously reported values for the tensile moduli of human and other animal cartilages determined from uniaxial tension experiments. This study provides the first available data for material properties and fixed charge density in cartilage from the guinea pig knee and suggests a promising method for tracking changes in cartilage mechanics in small animal models of degeneration.     

The size and shape of human and bovine antithrombin III.

Human and bovine antithrombin, purified by affinity chromatography on heparin-agarose, have been characterized with regard to chemical composition, size, shape and conformation. Both preparations were found to contain several active components of identical or similar size but different electrical charge. Amino acids and carbohydrate analyses revealed striking similarities between human and bovine antithrombin, while immunological analyses failed to demonstrate any cross-reactivity. The molecular weights were determined by sedimentation equilibrium to be 58 000 for human and 56 000 for bovine antithrombin. The small molecular weight difference suggested by these values was verified by several empirical methods of molecular weight estimation. Hydrodynamic measurements indicated that the two proteins have similar molecular shapes, both of which are slightly more extended that that of typical globular proteins. The internal folding of the two polypeptide chains is also similar, as evidenced by the identity of the far-ultraviolet circular dichroism spectra. Specifically, these analyses suggested a low alpha-helix content of both proteins. In conclusion, the marked structural similarity of human and bovine antithrombin indicates that the two proteins may also exhibit extensive functional similarities in the binding of heparin and the inhibition of various coagulation factors.     

No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids

The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.     

Interactions of ciprofloxacin with DPPC and DPPG: fluorescence anisotropy, ATR-FTIR and 31P NMR spectroscopies and conformational analysis

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.     

Crystal structure of a human Mob1 protein: toward understanding Mob-regulated cell cycle pathways

The Mob protein family comprises a group of highly conserved eukaryotic proteins whose founding member functions in the mitotic exit network. At the molecular level, Mob proteins act as kinase-activating subunits. We cloned a human Mob1 family member, Mob1A, and determined its three-dimensional structure by X-ray crystallography. The core of Mob1A consists of a four-helix bundle that is stabilized by a bound zinc atom. The N-terminal helix of the bundle is solvent exposed and together with adjacent secondary structure elements forms an evolutionarily conserved surface with a strong negative electrostatic potential. Several conditional mutant alleles of S. cerevisiae MOB1 target this surface and decrease its net negative charge. Interestingly, the kinases with which yeast Mob proteins interact have two conserved basic regions within their N-terminal lobe. Thus, Mob proteins may regulate their target kinases through electrostatic interactions mediated by conserved charged surfaces.     

The tensile properties of the cartilage of human femoral condyles related to the content of collagen and glycosaminoglycans

A method is described of measuring the tensile stiffness and fracture stress of human femoral condylar cartilage in planes parallel to and at increasing depth below the articular surface. The axis of tension was either parallel or perpendicular to the predominant collagen fibre direction in the superficial zone. Specimens were analysed for their collagen and glycosaminoglycan contents and partial correlation coefficients were determined between the tensile properties and each of the chemical constituents.The correlations between the tensile properties and the collagen content of specimens oriented parallel to the collagen fibre direction was statistically significant in the superficial zone but the significance level decreased with increasing depth. In specimens which were oriented perpendicularly to the collagen fibre direction the correlations between the above variables were less significant.There was no significant correlation between the tensile properties and the glycosaminoglycans in cartilage.Visibly normal specimens from the superficial layer which were situated adjacent to visibly degenerate cartilage were weaker and less stiff than specimens situated on normal joints or remote from visibly degenerate cartilage. Such differences decreased with depth below articular surface and were greater in parallel-oriented specimens.     

Altered patterns and synthesis of extracellular matrix macromolecules in early osteoarthritis.

The synthesis and contents of extracellular non-collagenous matrix macromolecules was studied in early and late human osteoarthritic (OA) cartilage obtained at surgery for sarcomas in the lower extremities (normal and early OA) or for total knee replacement (late stage OA). The early OA samples were those that had some fibrillation in the joint by visual examination. One group had fibrillation in the area sampled and the other group had no fibrillation. Cartilage was taken from the same topographical area on the medial femoral condyle in all the samples, labeled with [3H]leucine and [35S]sulfate for 4 h at 37 degrees C and extracted with 4 M guanidine-HCl. Analysis of the extracts showed that the total amount of proteoglycans relative to hydroxyproline content was higher in the early and late OA than in the normal cartilage. These proteoglycans showed a relatively lower [35S]sulfate incorporation into GAG chains and a higher [3H]leucine incorporation. The pattern of newly synthesized proteins was altered similarly in early and late OA. Notably, synthesis of cartilage oligomeric matrix protein (COMP), fibronectin, and cartilage intermediate layer protein (CILP) was increased, also reflected in their abundance as determined by enzyme-linked immunosorbent assay (ELISA). Collagen synthesis appeared significantly increased only in the late stage OA. The observed altered composition and pattern of biosynthesis indicate that the joint undergoes metabolic alterations early in the disease process, even before there is overt fibrillation of the tissue. The early OA samples studied appear to represent two distinct groups of early lesions in different stages of the process of cartilage deterioration as shown by their differences in relative rates of synthesis and abundance of proteins.     

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 2. Potentiometric titration of the native apo-A-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin.

A comparison of the ionization behaviour of the human apoA-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. These data taken together with the results of calorimetric and titration experiments with the apoA-I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251-257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.