Well-defined, non-native protein structures of low stability have been increasingly observed as intermediates in protein folding or as equilibrium structures populated under specific solvent conditions. These intermediate structures, frequently referred to as molten globule states, are characterized by the presence of secondary structure, a lack of significant tertiary contacts, increased hydrophobicity and partial specific volume as compared to native structures, and low cooperativity in thermal unfolding. The present study demonstrates that under acidic conditions (pH less than 3) the antibody MAK33 can assume a folded stable conformation. This A-state is characterized by a high degree of secondary structure, increased hydrophobicity, a native-like maximum wavelength of fluorescence emission, and a tendency toward slow aggregation. A prominent feature of this low-pH conformation is the stability against denaturant and thermal unfolding that is manifested in highly cooperative reversible phase transitions indicative of the existence of well-defined tertiary contacts. These thermodynamic results are corroborated by the kinetics of folding from the completely unfolded chain to the alternatively folded state at pH 2. The given data suggest that MAK33 at pH 2 adopts a cooperative structure that differs from the native immunoglobulin fold at pH 7. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its extraordinary structural stability that is characteristic for native protein structures.

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and the ¶Institut fü r Physikalische Chemie, Schlossplatz 7, Universitä t Mü nster, 48149 Mü nster, GermanyWe have recombinantly expressed a soluble form of human ␣ 2 ␤ 1 integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble ␣ 2 ␤ 1 integrin binds to its collagen ligands the same way as the wildtype ␣ 2 ␤ 1 integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrinactivating antibody. However, it does not bind to rhodocytin, a postulated agonist of ␣ 2 ␤ 1 integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble ␣ 2 ␤ 1 integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble ␣ 2 ␤ 1 integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble ␣ 2 ␤ 1 integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to ␣ 2 ␤ 1 integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with ␣ 2 ␤ 1 integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to ␣ 2 ␤ 1 integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking ␣ 2 ␤ 1 integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, ␣ 2 ␤ 1 integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, ␣ 2 ␤ 1 integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit ␣ 2 ␤ 1 integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.Integrins are cell adhesion molecules that consist of two noncovalently associated subunits, ␣ and ␤ (for review see Refs. 1 and 2). The subfamily of integrins sharing the ␤ 1 subunit are well known receptors for extracellular matrix molecules, such as collagens, laminins, and fibronectin. The subfamily of ␤ 3 subunit containing cytoadhesins comprise the platelet integrin, ␣ IIb ␤ 3 which binds fibrinogen/fibrin (3) and the vitronectin receptor ␣ V ␤ 3 . The latter ones, along with several ␤ 1 integrin, such as the fibronectin receptor ␣ 5 ␤ 1 integrin, recognize a linear arginyl-glycyl-aspartyl sequence within their respective ...

The role carbohydrate moieties play in determining the structure and energetics of glycolipid model membranes has been investigated by small- and wide-angle X-ray scattering, differential scanning densitometry (DSD), and differential scanning microcalorimetry (DSC). The dependence of a variety of thermodynamic and structural parameters on the stereochemistry of the OH groups in the pyranose ring and on the size of the sugar head group has been studied by using an homologous series of synthetic stereochemically uniform glyceroglycolipids having glucose, galactose, mannose, maltose, or trimaltose head groups and saturated ether-linked alkyl chains with 10, 12, 14, 16, or 18 carbon atoms per chain. The combined structural and thermodynamic data indicate that stereochemical changes of a single OH group in the pyranose ring can cause dramatic alterations in the stability and in the nature of the phase transitions of the membranes. The second equally important determinant of lipid interactions in the membrane is the size of the head group. A comparison of lipids with glucose, maltose, or trimaltose head groups and identical hydrophobic moieties has shown that increasing the size of the neutral carbohydrate head group strongly favors the bilayer-forming tendency of the glycolipids. These experimental results provide a verification of the geometric model advanced by Israelachvili et al. (1980) [Israelachvili, J. N., Marcelja, S., & Horn, R. G. (1980) Q. Rev. Biophys. 13, 121-200] to explain the preferences lipids exhibit for certain structures. Generally galactose head groups confer highest stability on the multilamellar model membranes as judged on the basis of the chain-melting transition. This is an interesting aspect in view of the fact that galactose moieties are frequently observed in membranes of thermophilic organisms. Glucose head groups provide lower stability but increase the number of stable intermediate structures that the corresponding lipids can adopt. Galactolipids do not even assume a stable intermediate L alpha phase for lipids with short chain length but perform only Lc----HII transitions in the first heating. The C2 isomer, mannose, modifies the phase preference in such a manner that only L beta----HII changes can occur. Maltose and trimaltose head groups prevent the adoption of the HII phase and permit only L beta----L alpha phase changes. The DSD studies resulted in a quantitative estimate for the volume change associated with the L alpha----HII transition of 14-Glc. The value of delta v = 0.005 mL/g supports the view that the volume difference between L alpha and HII is minute.(ABSTRACT TRUNCATED AT 400 WORDS)

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Detailed thermodynamic and spectroscopic studies were carried out on the ColE1-ROP protein in order to establish a quantitative basis for the contribution of noncovalent interactions to the stability of four-helix-bundle proteins. The energetics of both heat- and GdnHCl-induced denaturation were measured by differential scanning microcalorimetry (DSC) and/or by following the change in circular dichroism in the far-UV range. Sedimentation equilibrium analyses were performed to characterize the state of aggregation of the protein. No intermediate species could be detected during thermal unfolding of the dimer in the absence of GdnHCl. Under these conditions ROP unfolding exhibits a strict two-state behavior. The thermodynamic parameters for the reaction N2<->2D are delta HD = 580 +/- 20 kJ.(mol of dimer)-1, delta Cp = 10.3 +/- 1.3 kJ.(mol of dimer)-1.K-1, and Tm = 71.0 +/- 0.5 degrees C. The corresponding Gibbs energy change of unfolding is delta GD degree = 71.7 kJ.(mol of dimer)-1 at 25 degrees C and pH 6. In the presence of 2.5 M GdnHCl, however, ROP dissociates into monomers at elevated temperatures, as the loss of the concentration dependence of Tm and the decreased molecular weight demonstrate. The corresponding transition parameters are delta HD (2.5 M GdnHCl) = 130 +/- 10 kJ.(mol of monomer)-1 and Tm = 51.6 +/- 0.3 degrees C. Isothermal unfolding studies at 19 degrees C using GdnHCl as denaturant yielded a Gibbs energy change of unfolding of 22.4 kJ.(mol of monomer)-1. This extrapolated value is 38% lower than the corresponding delta GD degree value of 35.85 kJ.(mol of monomer)-1 calculated from thermal unfolding for the monomer in the absence of GdnHCl, where the protein is known to be a dimer. These results suggest that subunit interactions are an important source of stabilization of the native four-helix-bundle structure of ROP.

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The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)