HPr as a model protein in structure, interaction, folding and stability studies.

The small size and lack of disulphide bonds or cofactors in the Histidine-containing phosphocarrier protein (HPr) makes it an attractive system with which to study structure, interaction to its enzymatic partners, and its stability and folding. Here we give an overview on the immense work that has been performed on this protein and we will show that HPr has been widely used as a model protein to study important aspects in modern Structural Biology.

A thermodynamic analysis of a family of small globular proteins: SH3 domains.

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

Expression and characterization of the intact N-terminal domain of streptokinase.

Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero-Lara F et al., 1996, Protein Sci 5:2583-2591). The N-terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N-terminal region of the protein, residues 1-146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein.

Analysis of the interactions between streptokinase domains and human plasminogen.

The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.

Scanning calorimetry and Fourier-transform infrared studies into the thermal stability of cleaved bacteriorhodopsin systems.

Differential scanning calorimetry and Fourier-transform infrared spectroscopy have been used to characterize the thermal stability of bacteriorhodopsin (BR) cleaved within different loops connecting the helical rods. The results are compared to those of the native protein. We show that the denaturation temperature and enthalpy of BR cleaved at peptide bond 71-72 or 155-156 are lower than those of the intact protein, and that these values become even lower for the BR cleaved at both peptide bonds. The effect of cleavage on the denaturation temperature and enthalpy values seems to be additive as has been previously suggested [Khan, T. W., Sturtevant, J. M., & Engelman, D. M. (1992) Biochemistry 31, 8829]. The thermal denaturation of all the samples was irreversible and scan-rate dependent. When cleaved at the 71-72 bond BR follows quantitatively the predictions of the two-state kinetic model at pH 9.5, with an activation energy of 374 kJ/mol, similar to that of native BR. Calorimetry experiments with different populations of intact and cleaved BR provide direct evidence for some intermolecular cooperativity upon denaturation. The denatured samples maintain a large proportion of alpha helices and beta structure, a fact which seems to be related to their low denaturation enthalpy as compared to that of water-soluble, globular proteins.

Thermal stability of the three domains of streptokinase studied by circular dichroism and nuclear magnetic resonance.

Streptococcus equisimilis streptokinase (SK) is a single-chain protein of 414 residues that is used extensively in the clinical treatment of acute myocardial infarction due to its ability to activate human plasminogen (Plg). The mechanism by which this occurs is poorly understood due to the lack of structural details concerning both molecules and their complex. We reported recently (Parrado J et al., 1996, Protein Sci 5:693-704) that SK is composed of three structural domains (A, B, and C) with a C-terminal tail that is relatively unstructured. Here, we report thermal unfolding experiments, monitored by CD and NMR, using samples of intact SK, five isolated SK fragments, and two two-chain noncovalent complexes between complementary fragments of the protein. These experiments have allowed the unfolding processes of specific domains of the protein to be monitored and their relative stabilities and interdomain interactions to be characterized. Results demonstrate that SK can exist in a number of partially unfolded states, in which individual domains of the protein behave as single cooperative units. Domain B unfolds cooperatively in the first thermal transition at approximately 46 degrees C and its stability is largely independent of the presence of the other domains. The high-temperature transition in intact SK (at approximately 63 degrees C) corresponds to the unfolding of both domains A and C. Thermal stability of domain C is significantly increased by its isolation from the rest of the chain. By contrast, cleavage of the Phe 63-Ala 64 peptide bond within domain A causes thermal destabilization of this domain. The two resulting domain portions (A1 and A2) adopt unstructured conformations when separated. A1 binds with high affinity to all fragments that contain the A2 portion, with a concomitant restoration of the native-like fold of domain A. This result demonstrates that the mechanism whereby A1 stimulates the plasminogen activator activities of complementary SK fragments is the reconstitution of the native-like structure of domain A.

The thermodynamics of association and unfolding of the 205-316 C-terminal fragment of thermolysin.

The 205-316 C-terminal fragment of thermolysin has been studied by differential scanning calorimetry at pH values 2.5, 3.0, 3.5, 4.0 and 5.0 and at a constant ionic strength of 130 mM. The thermal unfolding of the fragment occurs at thermodynamic equilibrium under our experimental conditions. The effect of sample concentration at the different pH values on the calorimetric traces is consistent with a monomer-dimer equilibrium of the folded fragment, which undergoes thermal unfolding into individual fragments. Equilibrium sedimentation experiments at 10 degrees C and different pH values confirm the presence of the association equilibrium and provide the value of the dimerization constants. The global analysis of the calorimetric, heat capacity curves has been carried out by a multidimensional fitting to the model N2<-->2N<-->2U. The analysis leads to a complete thermodynamic characterization of both the association and unfolding processes of the fragment. The resulting thermodynamic functions suggest a partially unfolded structure for both the monomeric and dimeric fragment, as well as a conformational change linked to the association process. Our results are discussed in terms of the structural information currently available and compared with the energetics of unfolding of the shorter 255-316 dimeric C-terminal fragment of thermolysin (Conejero-Lara, F., De Filippis, V., Fontana, A. and Mateo, P.L. (1994) FEBS Lett. 344, 154-156). The presence of the additional 50 residues increases the relative population of the 205-316 monomeric fragment versus that of the 255-316 fragment.

Heat and cold denaturation of beta-lactoglobulin B.

The thermal denaturation of bovine beta-lactoglobulin B was investigated by high-sensitivity differential scanning microcalorimetry between pH 1.5 and 3.0 in 20 mM phosphate buffer. The process was found to be a reversible, two-state transition. Progressive addition of guanidine hydrochloride at pH 3.0 leads to the appearance of a low-temperature calorimetric endotherm, corresponding to the cold renaturation of the protein. Circular dichroism experiments have confirmed the low and high temperature denaturation processes, and have shown some structural differences between both denatured states of beta-lactoglobulin B.