Characteristics and properties of the unfolding free energy change, delta G degrees N-U, as determined by the linear extrapolation method are assessed for the unfolding of phenylmethanesulfonyl chymotrypsin (PMS-Ct). Difference spectral measurements at 293 nm were used to define PMS-Ct unfolding brought about with guanidinium chloride, urea, and 1,3-dimethylurea. All three denaturants were shown to give identical extinction coefficient differences (delta epsilon N-U) between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of delta epsilon N-U on denaturant supports the linear extension of pre- and postdenaturational base lines into the transition zone, allowing evaluation of unfolding equilibrium constants based on the two-state assumption. An expression, based on the linear extrapolation method, was used to provide estimates of delta G degrees N-U for the three denaturants using nonlinear least-squares fitting of the primary data, delta epsilon versus [denaturant]. The three delta G degrees N-U values were identical, within error, suggesting that the free energy change is a property of the protein system and independent of denaturant. It is suggested that the error in delta G degrees N-U determined from use of the linear extrapolation method is significantly larger than commonly reported in the literature.

Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS)

The enthalpy change (AM) accompanying the a-helix to random coil transition in water has been determined calorimetrically for a 50-residue peptide of defined sequence that contains primarily alanine. The enthalpy of helix formation is one of the basic parameters needed to predict thermal unfolding curves for peptide helices and it provides a starting point for analysis of the peptide hydrogen bond. The experimental uncertainty in AM reflects the fact that the transition curve is too broad to measure in its entirety, which precludes fitting the baselines directly. A lower limit for All ofunfolding, 0.9 kcal/mol per residue, is given by assuming that the change in heat capacity (ACp) is zero, and allowing the baseline to intersect the transition curve at the lowest measured Cp value.Use of the van't Hoff equation plus least-squares fitting to determine a more probable baseline gives AH = 1.3 kcal/mol per residue. Earlier studies of poly(L-lysine) and poly(Lglutamate) have given 1.1 kcal/mol per residue. Those investigations, along with our present result, suggest that the side chain has little effect on AH. The possibility that the peptide hydrogen bond shows a correspondingly large Al, and the implications for protein stability, are discussed.Although it has the smallest side chain except for glycine, alanine has one of the highest helix propensities (1-4) of the amino acids in the genetic code: alanine-rich peptides as short as 16 residues form isolated a-helices in water (1). This fact suggests that the a-helix is an intrinsically stable structure in water and that larger side chains, as well as polar side chains, more often detract from helix stability than add to it. Studies with model compounds (5-8) have given differing estimates of the stability of the amide hydrogen bond in water but agree that competing hydrogen bonds to water drastically limit the stability of the amide hydrogen bond. Measurement of the enthalpy change (All) and the heat capacity change (ACp) for alanine helix formation should provide important information about the energetics of the peptide hydrogen bond, which is one of the fundamental constants of protein stability. We expect that the temperature-independent component of AH should reflect the peptide hydrogen bond and van der Waals contacts, and the hydrophobic interactions will be reflected in ACp.We report the calorimetric measurement of AH for a-helix formation by a 50-residue peptide, I, whose sequence is Ac-Y(AEAAKA)8F-NH2.The results give the enthalpy of formation of a monomeric helix of defined sequence and length. Short peptides give broad thermal unfolding transition curves and small heats of unfolding, whereas long polypeptides are difficult to synthesize and purify. Compound I represents a compromise between these conflicting considerations. Synthesis of I began soon after the discovery that the sequence AEAAK, used as a repeating unit, forms stable helices when there are three repeats (9). The possible ion pairs formed by the Glu-, Lys' residues with an i, i + 3 s...

Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The linear extrapolation method was used to evaluate the unfolding free energy changes (delta G degrees N-U) for phenylmethanesulfonyl chymotrypsin (PMS-Ct) at pH 6.0. The nonlinear least-squares fits of difference spectral data using urea and guanidinium chloride as denaturants gave identical values for delta G degrees N-U and delta epsilon degrees U, the latter being extinction coefficient differences between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of these parameters from the nature of solvent suggests strongly that they are characteristic properties of the protein alone. The delta G degrees N-U data at pH 6.0 and 4.0, which differ by more than 100-fold in stability of the protein, were incorporated into a thermodynamic cycle involving free energy changes for titration of native and unfolded PMS-Ct from pH 4.0 to 6.0. The purpose of the cycle was to test whether delta G degrees N-U obtained by use of the linear extrapolation method exhibits the characteristics required of a thermodynamic function of state. Within error, the thermodynamic cycle was found to accommodate the delta G degrees N-U quantities obtained at pH 4.0 and 6.0 for PMS-Ct.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which infects a wide range of intermediate hosts that include different species of birds and mammals, including humans. The tachyzoites, the rapid multiplying form of the parasite, can invade and replicate within all nucleated cells and, if left unchecked by the immune system, causes extensive tissue damage and death of the intermediate host (11). Resistance to acute infection with T. gondii in the murine model is highly dependent on endogenous gamma interferon (IFN-␥) (11,12,37,43). Soon after initial infection in the intermediate host, T. gondii tachyzoites trigger the synthesis of interleukin-12 (2, 12, 13, 23, 34) and other costimulatory cytokines (13,17,22,23), which initiate the synthesis of IFN-␥ by NK cells (13, 23) and CD4 ϩ CD8 Ϫ ␣␤ ϩ T lymphocytes (12). IFN-␥ combined with tumor necrosis factor alpha will activate macrophages to produce high levels of reactive nitrogen intermediates (RNI) that are involved in the control of parasite replication (1, 26). However, RNI is only one of the IFN-␥-inducible mechanisms involved in the control of tachyzoite replication, and mice treated with inducible nitric oxide synthase (iNOS) inhibitor (20) or deficient in iNOS (38) are relatively more resistant than mice treated with neutralizing antibodies to IFN-␥ (12) or deficient in IFN-␥ or IFN-␥ receptor (10, 37). Thus, additional effector mechanisms induced by IFN-␥ and active during early experimental infection with T. gondii in the mouse model still have to be defined (11,21).Indoleamine 2,3-dioxygenase (INDO) is an enzyme that catalyzes the initial rate-limiting step of tryptophan (Trp) catabolism to N-formylkynurenine and kynurenine (Kyn) (21, 44). Many human cell lines express INDO upon stimulation with IFN-␥. Restriction of available Trp due to degradation by INDO leads to the control of various intracellular pathogens, including T. gondii, in both nonprofessional phagocytic cells (NPPC) and professional phagocytic cells (PPC) (4,5,7,8,28,29,32,39,45). In the absence of Trp, an essential amino acid for T. gondii, parasite growth also becomes restricted (32,36,41). In fact, in human NPPC the induction of INDO appears to be the main mechanism by which IFN-␥ controls the intracellular replication of T. gondii tachyzoites (4,5,7,8,29,32).The INDO activity and the Trp-Kyn metabolic pathway can be induced in murine tissues under various conditions (21,27,35,36). However, it has been difficult to demonstrate the role of INDO and Trp degradation in the control of tachyzoite replication in cell lines of mouse origin (18,19,40). In addition, no information is available about the induction of the Trp-Kyn metabolic pathway and its possible role in the restriction of parasite replication during in vivo experimental infection with T. gondii. In the present study, we evaluated the induction of INDO mRNA, Trp degradation, and Kyn formation during infection with T. gondii. Our results show that during the early stage of infection with T. gondii in the mouse ...

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In this study, we carried out a comparative analysis between two classical methodologies to prospect residue contacts in proteins: the traditional cutoff dependent (CD) approach and cutoff free Delaunay tessellation (DT). In addition, two alternative coarse-grained forms to represent residues were tested: using alpha carbon (CA) and side chain geometric center (GC). A database was built, comprising three top classes: all alpha, all beta, and alpha/beta. We found that the cutoff value at about 7.0 A emerges as an important distance parameter. Up to 7.0 A, CD and DT properties are unified, which implies that at this distance all contacts are complete and legitimate (not occluded). We also have shown that DT has an intrinsic missing edges problem when mapping the first layer of neighbors. In proteins, it may produce systematic errors affecting mainly the contact network in beta chains with CA. The almost-Delaunay (AD) approach has been proposed to solve this DT problem. We found that even AD may not be an advantageous solution. As a consequence, in the strict range up to 7.0 A, the CD approach revealed to be a simpler, more complete, and reliable technique than DT or AD. Finally, we have shown that coarse-grained residue representations may introduce bias in the analysis of neighbors in cutoffs up to 6.8 A, with CA favoring alpha proteins and GC favoring beta proteins. This provides an additional argument pointing to the value of 7.0 A as an important lower bound cutoff to be used in contact analysis of proteins.