Sedimentation equilibrium and low-angle laser-light scattering were used to determine the molar mass of the glycoprotein moieties in the complexes of sodium dodecyl sulphate with the human platelet membrane glycoproteins IIb (GPIIb), IIIa (GPIIIa), and the α (GPIIbα) and β (GPIIbβ) subunits of GPIIb. The values obtained by both procedures, except those for GPIIb, agree within experimental error with those calculated from their chemical composition: GPIIbα (114,000 g mol-1), GPIIbβ (22,200 g mol-1), and GPIIIa (91,500 g mol-1). The molar mass of GPIIb determined by light scattering (142,000 g mol-1) and sedimentation equilibrium at different solvent densities (134,000 g mol-1) also agree, within experimental error, with the values calculated either from its chemical composition (136,500 g mol-1) or from the sum of the molar masses of its subunits. However the molar mass determined by sedimentation equilibrium at constant solvent density, is consistently underestimated (116,000 g mol-1).

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.

LytA amidase is the best known bacterial autolysin. It breaks down the N-acetylmuramoyl-L-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae and requires the presence of choline residues in the cell-wall teichoic acids for activity. Genetic experiments have supported the hypothesis that its 36-kDa chain has evolved by the fusion of two independent modules: the NH-terminal module, responsible for the catalytic activity, and the COOH-terminal module, involved in the attachment to the cell wall. The structural organization of LytA amidase and of its isolated COOH-terminal module (C-LytA) and the variations induced by choline binding have been examined by differential scanning calorimetry and analytical ultracentrifugation. Deconvolution of calorimetric curves have revealed a folding of the polypeptide chain in several independent or quasi-independent cooperative domains. Elementary transitions in C-LytA are close but not identical to those assigned to the COOH-terminal module in the complete amidase, particularly in the absence of choline. These results indicate that the NH-terminal region of the protein is important for attaining the native tertiary fold of the COOH terminus. Analytical ultracentrifugation studies have shown that LytA exhibits a monomer dimer association equilibrium, through the COOH-terminal part of the molecule. Dimerization is regulated by choline interaction and involves the preferential binding of two molecules of choline per dimer. Sedimentation velocity experiments give frictional ratios of 1.1 for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that deviated from that of globular rigid particles. When considered together, present results give evidence that LytA amidase might be described as an elongated molecule consisting of at least four domains per subunit (two per module) designated here in as N1, N2, C1, and C2. Intersubunit cooperative interactions through the C2 domain in LytA dimer occur under all experimental conditions, while C-LytA requires the saturation of low affinity choline binding sites. The relevance of the structural features deduced here for LytA amidase is examined in connection with its biological function.

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.

Differential scanning calorimetry (DSC) has been employed to characterize the thermal denaturation of CPL1 lysozyme and its isolated fragments in the absence and presence of choline. The heat capacity function of CPL1 lysozyme shows two peaks with Tm values of 43.5 and 51.4 degrees C. At saturating concentrations of choline the second transition disappears, and the Tm is shifted to higher temperatures. The DSC thermogram of the C-CPL1 protein corresponding to the carboxyl-terminal domain of CPL1 lysozyme has a single peak with a Tm of 42.9 degrees C. The effect of choline is very similar to that observed for the whole CPL1 lysozyme. The NH2-terminal fragment obtained by proteolytic digestion shows a Tm of 52 degrees C, close to that of 51.4 degrees C found for the second transition of CPL1, and choline does not affect the Tm nor the denaturation enthalpy. These data suggest that choline is bound to the COOH-terminal domain of the protein. Deconvolution of the excess heat capacity curve of the CPL1 lysozyme shows that the data can be fitted to two two-state independent transitions. The analysis of the DSC curves showed that the NH2-terminal unfolding enthalpy steadily decreases with increasing concentrations of choline. These results indicate that, under saturating concentrations of choline, whole CPL1 could unfold as a single cooperative unit.

The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the αIIb subunit (GPIIb, 136 kDa) and the β3 subunit (GPIIIa, 92 kDa), serves as the fibrinogen receptor at the surface of activated platelets. The degree of dissociation of the GPIIb/IIIa heterodimer (s°20*, 8.9 S) into its constituent glycoproteins (GPIIb, 5.8 S; and GPIIIa, 3.9 S) has been assessed by analytical ultracentrifugation in Triton X100 buffers, and its Ca2+- and temperature-dependence correlated with Ca2+-binding to GPIIb/IIIa and its temperature dependence. At 21°C half-maximal dissociation of GPIIb/IIIa occurs at 5.5 ± 2.5 × 10−8 M Ca2+, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kd1 8 ± 3 × 10−8 M) (Rivas and González-Rodríguez, 1991) and much lower than the Kd of the 3.4 medium affinity Ca-binding sites (Kd2 4 ± 1.5 × 10−5 M), which seems to demonstrate that the stability of the heterodimer in solution at room temperature is regulated by the degree of saturation of the high-affinity Ca-binding site. At 4°C, the stability of the heterodimer is apparently Ca2+-independent, while at room and physiological temperatures (15–37°C) the degree of dissociation of the heterodimer is regulated by the degree of dissociation of the high- and medium-affinity Ca-binding sites, respectively. On increasing the Ca2+ concentration up to 1 × 10−4 M after dissociation in Triton X100 solutions, the reconstitution of the GPIIb/IIIa heterodimer depends on the time and temperature at which the dissociated heterodimer was maintained, being almost complete within the first 5–10 min at 37°C and within the first 1–2 h at 21°C. After this time, a time- and temperature-dependent irreversible autoassociation of GPIIb (covalent) and GPIIIa (non-covalent) occurs, which hinders both the isolation of permanently stable monoamers of GPIIb and GPIIIa and the reconstitution of the GPIIb/IIIa heterodimer in Triton X100 solutions.

The antitumour protein alpha-sarcin modifies the thermotropic behaviour of phospholipid vesicles. This has been studied by fluorescence depolarization measurements and differential scanning calorimetry. A surface protein-phospholipid interaction is detected by measuring the polarization degree of TMA-DPH-labelled vesicles. At the higher protein/lipid molar ratios studied, the alpha-sarcin-vesicles complexes exhibit different thermotropic behaviour depending on whether they are prepared above or below the Tm of the corresponding phospholipid. Labelling of the protein with photoactive phospholipids has also been considered. alpha-Sarcin penetrates the bilayer deep enough to be labelled with the photoactive group located at the C-12 of the fatty acid acyl chain of phospholipids forming vesicles.

Human platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca2+-dependent heterodimer, the integrin GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Below the critical micellar concentration of Triton X100 (TtX), the three glycoproteins do not bind appreciably to TtX and form association products of large size. The size-exclusion chromatographic patterns of GPIIb, GPIIIa and GPIIb/IIIa have been obtained at 0.2% TtX, and the molecular properties of the association products and monomer fractions have been determined by analysis of the detergent bound to the glycoproteins, laser-light scattering, sedimentation velocity, and electron microscopy (TEM). The monomer of the GPIIb-TtX complex was identified by the molecular mass (M) of the glycoprotein moiety (125 ± 15 kDa), the molecular size (9.5 ± 1.5 nm × 11 ±1.5 nm) and globular shape observed by TEM. It has a molecular mass (M*) of 197 ± 20 kDa, a sedimentation coefficient inf20 supo* of 5.8±0.1 S, a Stokes radius R infs sup* of 6.8±0.4 nm, and a frictional ratio f*/ infmin sup* of 1.7±0.14. The (GPIIb) n -TtX complexes are disulphide-bonded size-heterogeneous association products of GPIIb, tetramers being the smallest species found. GPIIIa has a greater propensity to self-associate than GPIIb, this tendency being lower below 1 mg GPIIIa/ml, 0.1 mM Ca2+, pH 9.0. The (GPIIIa) n -TtX complexes are noncovalent size-heterogeneous association products of GPIIIa, tetramers being the smallest form observed. The monomer of the GPIIIa-TtX complex was identified by the 103 ±15 kDa M determined for the glycoprotein moiety, and the 9 ± 1.5 nm × 10 ± 1.5 nm size and globular shape observed by TEM. It has a M* of 136 ± 15 kDa, a s inf20 supo* of 3.9 ± 0.3 S, a Rinfs/p* of 6.4 ± 0.5 nm, a f */f infmin sup* of 1.9 ± 0.3, and, when stored at pH 7.4, has a certain tendency to form filamentous association products (20–70 nm × 2–5 nm), as observed by TEM. The GPIIb/IIIa-TtX complex in 0.2% TtX/0.1 mM Ca2+ elutes as a single monomeric fraction, as deduced from the 210 ± 15 kDa M determined for its glycoprotein moiety and the 12 ± 1.5 nm × 14 ± 1.5 nm size of the globular forms observed by TEM. The GPIIb/IIIa-TtX complex has a M* of 315±20kDa, a s inf20 sup* of 8.9±0.2 S, a infs sup* of 7.4±0.2 nm, a f*/f infmin sup* of 1.5±0.12, and appears in the electron micrographs in multiple forms (filled globular, empty oval, head-two-tails, and bilobular shapes), which on lowering the TtX concentration tend to self-associate, forming a bundant rosettes below the TtX critical micellar concentration.

The interaction of fibrinogen with the mannose-specific lectins concanavalin A (ConA), its acetyl derivative (Ac-ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen B beta and gamma chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac-ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1 = 2.4 microM and Kd2 = 16.6 microM; n1 = n2 = 2) while only two moles of LcH are bound per mole of fibrinogen (Kd = 2.6 microM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac-ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac-ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectin-fibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac-ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen. The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5–7.5 at 25°C (μ = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5′-phosphoramidate are also reported at pH 6.9 (T = 25°C, μ = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5′-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: ΔG0 = −22 kJ mol−1, ΔH0 = −34 kJ mol−1 and ΔS0 = −40 J mol−1 K−1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: ΔG0 = −7.7 kJ mol−1, ΔH0 = −28 kJ mol−1 and ΔS0 = −68 J mol−1 K−1 and ΔG0 = −28 kJ mol−1, ΔHH0 = −10 kJ mol−1 and ΔSH0 = 61 J mol−1 K−1 respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.

Low frequency ultrasound irradiation of purified fibrinogen solutions produces a precipitation of protein and a loose on clottability due to molecular fragmentation, both depending in different ways on the irradiation dose. Precipitation seems to be closely related to free radical formation, the fragmentation being due mainly to the shearing forces appearing during cavitation. The high molecular weight fragments obtained by ultrasound irradiation of fibrinogen have been isolated by gel filtration and studied by disc gel electrophoresis and analytical ultracentrifugation. They show molecular properties similar to those of the fragments obtained by endopeptidase incubation and the protein degradation proceeds in a similar way. All that can be interpreted according to a trinodular model for the fibrinogen molecule, with two elongated fibrous zones more easily cleaved by mechanical forces and less compact parts of the molecule that are mostly constituted by the α-chains.

The high molecular weight fragments observed during tryptic digestion of bovine fibrinogen and the variation of their relative proportion with time has been studied. Separation of the different molecular species was carried out by gel filtration and the molecular weights of the isolated fragments were determined by sedimentation equilibrium and from their electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels. The fibrinogen is degraded by trypsin into distinct fragments, with molecular weights of 270 000, 170 000, 90 000 and 50 000, accompanied by a series of smaller fragments whose properties were not investigated. The relative proportion of the components was estimated from area measurements on scans of the stained gels obtained after electrophoresis in the presence of sodium dodecylsulfate/The relative concentration and the molecular weight of each component established its molar concentration in each of the digestion mixtures obtained after varying incubation times (1–60 min). These data were used for a kinetic analysis of the process. The kinetic model derived on the basis of the trinodular model of fibrinogen (see Appendix) gave a very good representation of all the experimental results.

Bacteriophage φ29 has been purified in large scale to homogeneity. The sedimentation and diffusion coefficients of the virus are (256 ± 2) × 10−13 sec and (8.8 ± 0.5) × 10−8 cm2 × sec−1, respectively. The value of the partial specific volume of φ29, obtained by pycnometry, is 0.61 ± 0.01 cm3 × g−1. From these values and the Svedberg equation, the molecular mass of φ29 is (18 ± 1) × 106 daltons. From the phosphorus and nitrogen content, and the amino acid and nucleotide composition, the DNA percentage in φ29 is 56 ± 2%. Other independent estimations of the molecular mass of φ29 are given.