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We recently reported that the ubiquitous, secreted protein clusterin has chaperone activity in vitro [Humphreys et al. (1999) J. Biol. Chem. 274, 6875-6881]. In this study, we demonstrate that clusterin (i) inhibits stress-induced precipitation of a very broad range of structurally divergent protein substrates, (ii) binds irreversibly via an ATP-independent mechanism to stressed proteins to form solubilized high molecular weight complexes, (iii) lacks detectable ATPase activity, (iv) when acting alone, does not effect refolding of stressed proteins in vitro, and (v) stabilizes stressed proteins in a state competent for refolding by heat shock protein 70 (HSP70). Furthermore, we show that, at physiological levels, clusterin inhibits stress-induced precipitation of proteins in undiluted human serum. Clusterin represents the first identified secreted mammalian chaperone. However, reports from others suggest that, at least under stress conditions, clusterin may be retained within cells to exert a protective effect. Regardless of the topological site(s) of action, the demonstration that clusterin can stabilize stressed proteins in a refolding-competent state suggests that, during stresses, the action of clusterin may inhibit rapid and irreversible protein precipitation and produce a reservoir of inactive but stabilized molecules from which other refolding chaperones can subsequently salvage functional proteins.

Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures. The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements. Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution. While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set. The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation. CBDcex forms an extensive beta-sheet structure with a beta-barrel fold. Titration with cellohexaose, [beta-D-glucopyranosyl-(1,4)]5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding. Analysis of the structure shows that these residues are adjacent in space and exposed to solvent. Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family.

The effect of internal motions on proton relaxation data in oligosaccharides has been investigated experimentally. 1H steady-state and transient NOEs together with 13C T1's have been measured at two magnetic field strengths. The existence of internal motions leads to additional modulations of the dipolar interaction between proton pairs, thus producing a range of spectral density functions for these interactions. As a result, it is possible to show that protons relaxing through fixed distances have a different ratio of relaxation parameters, acquired at 500 and 300 MHz, compared to those relaxing through fluctuating distances. This approach has been used to unequivocally establish for two disaccharides the existence of internal motions on the time scale of the overall tumbling.

The thermodynamic parameters that characterize the binding of wheat germ agglutinin isolectin I (WGA I) to the alpha 2-3 isomer of (N-acetylneuraminyl)lactose have been determined by 360-MHz proton nuclear magnetic resonance spectroscopy. The chemical exchange of the ligand between the free and bound sites resulted in a broadening and upfield shifting of the N-acetyl methyl resonance [Kronis, K.A., & Carver, J.P. (1985) Biochemistry (preceding paper in this issue)] which has allowed the determination of the equilibrium constant, KD, and the dissociation rate constant, kD. In this paper, the analysis of the temperature dependence of the KD values between 25.4 and 51.6 degrees C yielded equilibrium parameters indicative of a large entropy barrier to binding: delta H degree = -13.3 +/- 1.0 kcal mol-1 and delta S degree = -31.9 +/- 2.4 cal mol-1 K-1. The Arrhenius plot of the effect of temperature on the dissociation rate (kD) and the plot of 1n (kD/T) vs. 1/T indicated that the transition complex represented an unfavorable energy state compared to the dissociated molecules with an activation energy (EA) of +18.0 kcal mol-1 and enthalpy and entropy of dissociation (delta HD not equal to and delta SD not equal to) values of +17.4 +/- 0.3 kcal mol-1 and +13.4 +/- 1.2 cal mol-1 K-1, respectively. The driving force for this binding reaction is the large negative delta H degree with a small enthalpic barrier to association (delta HA = +4.1 kcal mol-1).(ABSTRACT TRUNCATED AT 250 WORDS)

A detailed analysis of the proton magnetic resonance spectral parameters for the anomeric and C2 hydrogen resonances of 63 different glycopeptides and oligosaccharides of known structure reveals a general method for the determination of the primary structure of glycopeptides for most currently known classes of structures. In particular, a two-dimensional display formed by plotting mannosyl C1-H vs. C2-H chemical shifts demonstrates that these pairs of values are sensitive to long-range perturbation by remote substitution by hexoses as well as to direct substitution effects. A total of 41 Cl-H/C2-H chemical shift clusters have been defined which characterize unique structural microenvironments. On the basis, the sequence and branching pattern for most structures can be derived. Corroborative evidence is obtained from an examination of the chemical shifts of the galactosyl and N-acetylglucosaminyl anomeric hydrogens as well as other features of the spectrum.

The carbohydrate attached at Asn-107 of the light chain of a human myeloma IgG1 kappa (Hom) was isolated and the structure determined by 1H NMR. Two oligosaccharides were found corresponding to mono- and disialylated forms of the bisected biantennary class of glycopeptides. Both structures had Fuc alpha 1-6 linked to the GlcNAc residue attached to Asn and NeuNAc residues linked alpha 2-6. Because of the unusual nature of these structures, the Asn-297 oligosaccharides of the same IgG were prepared from Fc fragments and heavy chains. Comparison of the structures of the latter glycopeptides with structures from the same site on a second human myeloma IgG1 kappa (Tem) showed them to be quite similar in that the majority of the structures were biantennary but not bisected. We suggest that the completely bisected nature of the light-chain oligosaccharides comes from a high level of activity of GlcNAc-T-III (the enzyme responsible for the attachment of the bisecting GlcNAc) in the cells producing the IgG. We suggest a mechanism for differential glycosylation between the Asn-107 and Asn-297 sites based on the stabilization of the Asn-297 oligosaccharide in a conformation with the torsional angle omega about the C5-C6 bond of the Man alpha 1-6 linkage equal to -60 degrees. It has previously been postulated that this conformation is not a substrate for GlcNAc-T-III [Brisson, J.-R., & Carver, J. P. (1983) Can. J. Biochem. 61, 1067-1078].(ABSTRACT TRUNCATED AT 250 WORDS)

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.

Sindbis virus membrane glycopeptides have ben purified in chemical quantities and their oligosaccharide structures analyzed by 1H NMR spectroscopy at 360 MHz. Interpretable spectra could be obtained with approximately 100 micrograms of oligosaccharide. Spectral analysis of the sialyl glycopeptides S1, S2, and S3 at high and low temperatures confirms their structures to be NANA alpha (2,3)Gal beta (1,4)-GlcNAc beta (1,2)Man alpha (1,6)-[NANA alpha (2,3)Gal beta (1,4)-GlcNAc beta (1,2)Man alpha (1,3)]-Man beta (1,4)GlcNAc beta (1,4)-[Fuc alpha-(1,6)]-GlcNAc beta 1-Asn. These are heterogeneous with respect to sialic acid (NANA). Spectra of two endo-beta-N-acetylglucosaminidase products of the S4 glycopeptides are reported. The interpretation of these spectra is consistent with Man5GlcNAc and Man7GlcNAc oligosaccharide structures. Their chemical shifts are essentially identical with those reported for ovalbumin glycopeptides of the same composition, with exception to the perturbations arising from their oligosaccharide nature.

Applications of ensemble averaging to the solution conformation of model compounds for N-linked glycans are further investigated. Specifically, the interpretation usually applied to observed values of J5,6', a parameter reflecting the rotameric distribution about C5-C6 bonds (torsion angle omega in 1,6 glycosidic linkages) in 6-O-substituted hexopyranosides, was found to be inconsistent with populations derived from potential energy calculations. However, agreement between observed and calculated, ensemble-averaged values of J5,6' was obtained and the distribution of omega rotamers reinterpreted. Values of J5,6' that were previously interpreted as indicative of equipartition between two rotamers in fact reflect a marked preference for one of them. Additional potential energy terms, previously absent from energy calculations, are introduced and shown to be without effect on interpretations of omega rotamer distributions. From comparisons with both NMR relaxation and scalar coupling constant data, it is concluded that a simple empirical algorithm, HSEA, calculating van der Waals, exo-anomeric, and (as appropriate) hydrogen-bonding terms, is best suited for describing the population distributions in solution for oligosaccharides and N-linked glycans.

Four glycopeptides have been purified by Dowex and Bio-Gel P2 chromatography from Pronase digests of hen ovalbumin. The high-resolution proton magnetic resonance spectra of these glycopeptides and various products of their enzymatic digestion have been obtained at 360 MHz. By use of information derived from the spectra of a number of model compounds, an unambiguous assignment of all C1-H and Man C2-H resonances in the spectra can be made. On this basis structures are proposed for the four glycopeptides which are identical with those structures previously deduced from destructive chemical methods.

The solution conformation of model compounds for the tri'-antennary and tetraantennary (six-arm) branch point of N-linked glycans has been determined through the use of chemical shift, relaxation, and nuclear Overhauser enhancement data. The object was to establish the conformation about the glycosidic linkages in the N-linked substructure GlcNAc(beta 1,6) [GlcNAc(beta 1,2)] Man(alpha)- by estimation of values for the appropriate glycosidic torsional angles. The GlcNAc(beta 1,6) linkage in a trisaccharide model compound was found to be constrained to a narrow rotameric subpopulation about the substituted Man C5-C6 bond (omega = -60 degrees) and a narrow range of possible phi - psi values. Free rotation about the Man C5-C6 bond was obstructed by unfavorable steric interactions between the GlcNAc(beta 1,6) and GlcNAc(beta 1,2) residues. A phi, psi value of 55 degrees, 190 degrees was found to be consistent with the NMR data for the GlcNAc(beta 1,6) linkage. However, the value of psi appears to be "virtual" in that the molecule is in equilibrium between two different values (90 degrees and 252 degrees). For the GlcNAc(beta 1,2) linkage, complete agreement between all the observed NMR parameters and all the calculated ensemble average values could only be obtained with a set of potential energy functions which included hydrogen bonding. Other choices of potentials yielded calculated values that disagreed with at least two of the observed quantities. As a result, we infer that an interresidue hydrogen bond is formed, and we find it to be between the GlcNAc(beta 1,2) ring oxygen and the Man C3 hydroxyl.(ABSTRACT TRUNCATED AT 250 WORDS)

In the accompanying paper (Bendiak et al., 1989), the separation of a series of oligosaccharides released from asparagine residues of fetuin was described. A series of NMR experiments, which included one- and two-dimensional nuclear Overhauser enhancement, two-dimensional correlation spectroscopy, and two-dimensional relayed-coherence spectroscopy, as well as permethylation analyses, established a Gal beta 1----3(NeuAc alpha 2----6)GlcNAc beta 1----4Man unit common to a series of purified structures. These oligosaccharides contained either three, four, or five glycosidically linked sialic acid residues. The NeuAc residue in alpha 2----6 linkage to GlcNAc gives rise to diagnostic chemical shift perturbations of particular proton signals in the oligosaccharides.

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.

The binding of three purified sialic acid containing oligosaccharides to two isolectins of wheat germ agglutinin (WGA I and WGA II) has been quantitated by measuring the broadening of a ligand resonance in the proton nuclear magnetic resonance (1H NMR) spectrum at 360 MHz. The ligands, isolated from bovine colostrum by using the procedure of Schneir and Rafelson [Schneir, M. L., & Rafelson, M. E., Jr. (1966) Biochim. Biophys, Acta 130, 1--11], were identified by 1H NMR as the alpha (2,3) and alpha (2,6) isomers of N-acetylneuraminyllactose, as well as the alpha (2,6) form of N,N'-diacetylneuraminyllactosamine. The dissociation constants, KD's, ranged from 0.7 to 10 mM (24 +/- 1 degree C). Two noteworthy features of WGA specificity emerge from an examination of the observed affinities: (1) both isolectins bind the alpha (2,3) isomer of N-acetylneuraminyllactose with higher affinity than the alpha (2,6) form and (2) WGA I binds two of the sialyloligosaccharides more tightly than does WGA II.

Equilibrium binding studies have been performed over a range of temperatures from 25.4 to 47.3 degrees C between wheat germ agglutinin isolectin I (WGA I) and the alpha 2-3 isomer of (N-acetylneuraminyl)lactose (NeuNAc alpha 2-3Gal beta 1-4G1c). Proton nuclear magnetic resonance spectroscopy at 360 MHz has been used to monitor titrations in this system under conditions where the fraction of total ligand which is bound is small, yet the fractional occupation of sites covers a wide range. Several of the ligand resonances, including the N-acetyl methyl and the axial and equatorial hydrogens at carbon 3 of the NeuNAc residue, are shifted and broadened in the presence of WGA due to chemical exchange between the free and bound environments. The lifetime broadening of the N-acetyl resonance at room temperature of a series of related sialyloligosaccharides has been previously used by us to measure binding affinities to two WGA isolectins [Kronis, K.A., & Carver, J.P. (1982) Biochemistry 21, 3050-3057]. In this paper we report the temperature dependence of the apparent bound shifts and the apparent bound line widths of the N-acetyl, H3a, and H3e peaks. The true bound shifts for the three resonances have been obtained from these data by using the equations derived by Swift and Connick [Swift, T.J., & Connick, R.E. (1962) J. Chem. Phys. 37, 307-320]. The total bound shifts, per monomer, were found to be -1.98, -4.0, and -0.8 ppm for the N-acetyl, the H3a, and the H3e resonances, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A 1H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the only ionizable group, Glu-18.

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].

In paramagnetic metmyoglobin, cyanomyoglobin (CNMb), and deoxymyoglobin, His-36 has a high pK (approximately 8), and the NMR titration behavior of the H-2 resonance is perturbed, due to the presence at low pH of a hydrogen bond with Glu-38, which is broken at high pH. The His-36 H-4 resonance shows no shift with pK approximately 8 because of two opposing chemical shift effects but monitors the titration of nearby Glu-36 (pK = 5.6). In diamagnetic derivatives [(carbon monoxy)myoglobin (COMb) and oxymyoglobin (oxyMb)], the titration behavior of His-36 H-2 and H-4 resonances is normalized (pK approximately 6.8). The very slight alkaline Bohr effect in sperm whale myoglobin (Mb) is interpreted in terms of the pK change of His-36 from deoxyMb to oxyMb and compensating pK changes in the opposite direction of other unspecified groups. In sperm whale COMb at 40 degrees C, the distal histidine (His-64) and His-97 have pK values of 5.0 and 5.9. The meso proton resonances remote from these groups do not show a titration shift, but the nearby gamma-meso proton (pK = 5.3) responds to titration of both histidines, and the upfield Val-68 methyl at -2.3 ppm (pK = 4.7) witnesses the titration of nearby His-64. At 20 degrees C, the latter resonance is reduced in size, and a second resonance occurs at -2.8 ppm, which is insensitive to pH and, hence, more remote from His-64. Both resonances arise from two conformations of Val-68 in slow equilibrium. In oxyMb at 20 degrees C, only the latter resonance is observed, presumably because of the steric restrictions imposed by the hydrogen bond between ligand and His-64 in oxyMb, which is not present in COMb. In oxyMb the pK of His-97 (5.6) is similar to that of the meso proton resonances (5.5) and to the pK of other pH-dependent processes, including the very small acid Bohr effect. It is likely that these processes are controlled by the titration of His-97.(ABSTRACT TRUNCATED AT 250 WORDS)

The solution conformations of synthetic methyl mannobiosides and a methyl mannotrioside containing alpha D(1-3) and alpha D(1-6) linkages have been determined through the use of 1H nuclear magnetic resonance techniques, namely, the nuclear Overhauser effect and proton relaxation time measurements. 3J(C,H) coupling constants, obtained from a compound enriched with 13C, were also used. The allowed conformations were found to be in agreement with those determined from potential energy calculations and crystal structures. The methyl mannotrioside is an analogue of a mannotriose unit which occurs naturally in the "core" of asparagine-linked glycopeptides and in an "arm" of high mannose N-linked glycopeptides.

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of the alpha(1-6)-linked moiety is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions and to draw conclusions about the behavior in solution of these molecules. For all classes, identical conformations were found for the 6-arm except for the torsional angle, omega, about the C5-C6 bond of the alpha 1-6 linkage. For high mannose and hybrid structures omega was found to be -60 degrees, for bisected biantennary complex structures omega was 180 degrees, and for complex biantennary structures averaging between -60 degrees and 180 degrees occurs.

Asparagine-linked oligosaccharides of the glycoprotein fetuin were isolated as reducing oligosaccharides after hydrazinolysis/re-N-acetylation/mild acid treatment of the Pronase-digested glycoprotein. The sialylated oligosaccharides were separated by high-performance liquid chromatography in two different systems, which resulted in greater than 35 fractions, comprising di-, tri-, tetra-, and pentasialylated oligosaccharides. The major components were isomeric structures comprising the tri- and tetrasialylated fractions. In this and the accompanying paper (Cumming et al., 1989), the structures of 10 of the major components of the tri-, tetra-, and pentasialylated oligosaccharide fractions are described. Separation protocols and three isolated structures having sialic acid linked only to galactose are presented in this paper.