Phosphorescence and optically detected magnetic resonance characterization of the environments of tryptophan analogues in staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment

Phosphorescence and optically detected magnetic resonance (ODMR) measurements are reported on the triplet states of the tryptophan analogues, 7-azatryptophan (7AW), 5-hydroxytryptophan (5HW), and 4-, 5-, and 6-fluorotryptophan (4FW, 5FW, 6FW), when incorporated at position 140 of wild-type Staphylococcal nuclease (7AW-nuclease, etc. ), positions 66 and 140 of its V66W mutant (7AW-V66W, etc.), and the deletion fragment of the latter, Delta 137-149 (7AW-V66W', etc.). These measurements point to the retention of protein structure at position 140 in each of the wild-type nuclease analogues. Substitution of the analogue at both tryptophan sites of V66W leads to structured sites with differentiated triplet-state properties for all analogues except 7AW-V66W, whose structure is destabilized. 5HW-V66W' is the only fragment that apparently lacks structure at position 66. All other V66W' analogues exhibit a structured environment at position 66 (4FW-V66W' was not studied), but in each case this site can be differentiated readily from the corresponding site in intact V66W. 7AW-V66W' is resolved by ODMR into two discrete structures with slightly differing zero field splittings (ZFS). Interaction of the protein with 5HW at position 66 of 5HW-V66W induces a 2-fold increase in the ZFS E parameter, which is reduced to its normal value upon formation of the fragment, 5HW-V66W'. Analogous effects occur for 5FW, but on a smaller scale.     

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure

A combination of circular dichroism spectroscopy, titration calorimetry, and optical melting has been used to investigate the association of the minor groove ligands netropsin and distamycin to the central A3T2 binding site of the DNA duplex d(CGCAAATTGGC).d(GCCAATTTGCG). For the complex with netropsin at 20 degrees C, a ligand/duplex stoichiometry of 1:1 was obtained with Kb approximately 4.3 x 10(7) M-1, delta Hb approximately -7.5 kcal mol-1, delta Sb approximately 9.3 cal K-1 mol-1, and delta Cp approximately 0. Previous NMR studies characterized the distamycin complex with A3T2 at saturation as a dimeric side-by-side complex. Consistent with this result, we found a ligand/duplex stoichiometry of 2:1. In the current study, the relative thermodynamic contributions of the two distamycin ligands in the formation of this side-by-side complex (2:1 Dst.A3T2) were evaluated and compared with the thermodynamic characteristics of netropsin binding. The association of the first distamycin molecule of the 2:1 Dst.A3T2 complex yielded the following thermodynamic profile: Kb approximately 3.1 x 10(7) M-1, delta Hb = -12.3 kcal mol-1, delta Sb = -8 cal K-1 mol-1, and delta Cp = -42 cal K-1 mol-1. The binding of the second distamycin molecule occurs with a lower Kb of approximately 3.3 x 10(6) M-1, a more favorable delta Hb of -18.8 kcal mol-1, a more unfavorable delta Sb of -34 cal K-1 mol-1, and a higher delta Cp of -196 cal K-1 mol-1. The latter term indicates an ordering of electrostricted and structural water molecules by the complexes. These results correlate well with the NMR titrations and are discussed in context of the solution structure of the 2:1 Dst.A3T2 complex.     

Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus comprises an N-terminal domain (residues 1-319), which is dimeric and forms tyrosyladenylate, and a C-terminal domain (residues 320-419), which binds the anticodon arm of tRNATyr. The N-terminal domain has the characteristic fold of the class I aminoacyl-tRNA synthetases. The unfolding of the N-terminal domain by urea at 25 degreesC under equilibrium conditions was monitored by its intensities of light emission at 330 and 350 nm, the ratio of these intensities, its ellipticity at 229 nm, and its partition coefficient, in spectrofluorometry, circular dichroism, and size-exclusion chromatography experiments, respectively. These experiments showed the existence of an equilibrium between the native dimeric state of the N-terminal domain, a monomeric intermediate state, and the unfolded state. The intermediate was compact and had secondary structure, and its tryptophan residues were partially buried. These properties of the intermediate and its inability to bind 1-anilino-8-naphthalenesulfonate showed that it was not in a molten globular state. The variation of free energy deltaG(H2O) and its coefficient m of dependence on the concentration of urea were, respectively, 13.8 +/- 0.2 kcal.mol-1 and 0.9 +/- 0.1 kcal.mol-1.M-1 for the dissociation of the native dimer and 13.9 +/- 0.6 kcal.mol-1 and 2.5 +/- 0.1 kcal.mol-1.M-1 for the unfolding of the monomeric intermediate.     

Cooperative folding of a protein mini domain: the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex

The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a tm of 54 degrees C. This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements. Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable alpha-helices. Chemical denaturation by guanidine hydrochloride is also cooperative with a delta GH2O of 3.1 kcal mol-1 at pH 8.0 and 25 degrees C. The chemical denaturation is broad with an m-value of 760 cal mol-1 M-1. psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold. A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine. This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a tm of only 31 degrees C. Chemical denaturations at 4 degrees C give an m-value of 740 cal mol-1 M-1, similar to the wild-type, but the stability delta GH2O is only 1.4 kcal mol-1. Both the wild-type and the mutant unfold at extremes of pH, but at 4 degrees C psbd41Asn is folded over a narrower pH range than the wild-type. Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to fold in the absence of disulfide bonds or metal or ligand-binding sites.     

Kinetic mechanism of a partial folding reaction. 1. Properties Of the reaction and effects of denaturants

The bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (Kd) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (kon = 1.8 x 10(7) M-1 s-1) and is very stable (Kd = 6 pM) at 10 degrees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol-1. The charge effect enters in the refolding reaction: increasing the salt concentration reduces kon dramatically and has little effect on koff. Urea and GdmCl destabilize the complex by decreasing kon and increasing koff. The slopes (m-values) of plots of ln Kd vs [cosolvent] are 0.75 +/- 0.04 and 2.8 +/- 0.3 kcal mol-1 M-1 for urea and GdmCl, respectively. The ratio mon/(mon + moff) is 0.54 +/- 0.04 for urea and 0.57 +/- 0.1 for GdmCl, where mon is the m-value for kon and moff is the m-value for koff, indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.     

Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride

The guanidine hydrochloride induced unfolding of the major fraction of ovalbumin (i.e. A1 which contains two phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectran and viscosity measurements. As judged by the intrinsic viscosity (3.9 ml/g), the native protein conformation is compact and globular. Difference spectral results showed extensive disruption of the native structure by guanidine hydrochloride with and without 0.1 M beta-mercaptoethanol were 31.1 and 27.0 ml/g. These and optical rotation results indicated that the denatured protein existed in a cross-linked random coil conformation in 6 M guanidine hydrochloride alone. Strikingly, in contrast to whole ovalbumin, the denaturation of its A1 fraction by guanidine hydrochloride was fully reversible and obeyed first-order kinetic law under different experimental condit ions of pH, temperature, and the denaturant concentration. The monotonic variation of deltaH for the unfolding of ovalbumin A1 by guanidine hydrochloride with temperature, the coincidence of the two transition curves obtained by measuring two independent properties (namely reduced viscosity and difference in light absorption at 288 nm (or 293 nm) as a function of the denaturant concentration, and finally the adherence of the unfolding as well as refolding reactions to first-order kinetic law suggested that the transition of ovalbumin. A1 can reasonably be approximated by a two-state mode. Analysis of the equilibrium data obtained at pH 7.0 and 25 degrees C according to Aune and Tanford (Aune, K.C.,and Tanford, C. (1969), Biochemistry 8, 4586) showed that 12 additional binding sites for the denaturant with an association constant of 1.12 were freshly exposed by the unfolding process and that the native protein was marginally more stable (approximately 6 kcal/mol) than its unfolded form even under native condition. The temperature dependence of the equilibrium constant for the unfolding of ovalbumin A1 by guanidine hydrochloride which was studied in the range 10-60 degrees C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: deltaH = 52 kcal/mol at 25 degrees C; deltaS = 153 cal deg-1 mol-1 at 25 degrees C; and delta Cp = 2700 +/- 400 cal deg-1 mol-1.     

Pressure denaturation of proteins: evaluation of compressibility effects

One of the key pieces of information from pressure denaturation experiments is the standard volume change for unfolding (Delta V(o)). The pressure dependence of the volume change, the standard compressibility change (Delta K(o)T), is typically assumed to be zero in the analysis of these experiments. We show here that this assumption can be incorrect and that the neglect of compressibility differences can skew the interpretation of experimental results. Analysis of experimental, variable-pressure NMR data for bovine pancreatic ribonuclease A in 2H2O at pH 2.0 and 295 K yielded the following statistically significant, non-zero values: Delta K(o) T = 0.015 +/- 0.002 mL mol-1 bar-1, Delta V(o) = -21 +/- 2 mL mol-1, and Delta G(o) = 2.8 +/- 0.3 kcal mol-1. The experimental protein stability is in good agreement with one (Delta G(o) = 2.5 kcal mol-1) determined independently for the same protein by calorimetry at atmospheric pressure under equivalent conditions [Makhatadze, G. I., Clore, G. M., and Gronenborn, A. M. (1995) Nat. Struct. Biol. 2, 852-855]. The positive value for Delta K(o)T indicates that the denatured form of ribonuclease A is more compressible than the native form; this is explained in terms of an interplay between the intrinsic compressibility of the protein and solvation effects. When the same data were fitted to a model that assumes a zero compressibility change, the Delta G(o) value of 4. 0 +/- 0.1 kcal mol-1 returned by the model no longer agreed with the independent measurement, and the Delta V(o) returned by the model was a very different -59 +/- 1 mL mol-1. By contrast, it was not possible to carry out a similar thermodynamic analysis of fluorescence spectroscopic data for the denaturation of staphylococcal nuclease to yield well-defined values of Delta G(o), Delta V(o), and Delta K(o)T. This limitation was shown by evaluation of synthetic data to be intrinsic to spectroscopic data whose analysis requires fitting of the plateaus at either side of the transition. Because NMR data do not have this requirement, they can be analyzed more rigorously.     

Conformational stability of muscle acylphosphatase: the role of temperature, denaturant concentration, and pH

The conformational stability (delta G) of muscle acylphosphatase, a small alpha/beta globular protein, has been determined as a function of temperature, urea concentration, and pH. A combination of thermally induced and urea-induced unfolding, monitored by far-UV circular dichroism, was used to define the conformational stability over a wide range of temperature. Through analysis of all these data, the heat capacity change upon unfolding (delta Cp) could be estimated, allowing the determination of the temperature dependence of the main thermodynamic functions (delta G, delta H, delta S). Thermal unfolding in the presence of urea made it possible to extend such thermodynamic analysis to examine these parameters as a function of urea concentration. The results indicate that acylphosphatase is a relatively unstable protein with a delta G(H2O) of 22 +/- 1 kJ mol-1 at pH 7 and 25 degrees C. The midpoints of both thermal and chemical denaturation are also relatively low. Urea denaturation curves over the pH range 2-12 have allowed the pH dependence of delta G to be determined and indicate that the maximum stability of the protein occurs near pH 5.5. While the dependence of delta G on urea (the m value) does not vary with temperature, a significant increase has been found at low pH values, suggesting that the overall dimensions of the unfolded state are significantly affected by the number of charges within the polypeptide chain. The comparison of these data with those from other small proteins indicates that the pattern of conformational stability is defined by individual sequences and not by the overall structural fold.     

Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Delta 137-149 fragment of the latter mutant. The guanidine-HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Delta 137-149 fragment of V66W) and at both positions (in V66W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wild-type nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-Azatryptophan is found to destabilize all three proteins. 4-Fluorotryptophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.     

Thermodynamic analysis of human plasma apolipoprotein C-1: high-temperature unfolding and low-temperature oligomer dissociation

Thermal and chemical unfolding of lipid-free apolipoprotein C-1 (apoC-1), a 6-kDa protein component of very low density and high-density lipoproteins, was analyzed by far-UV CD. In neutral 1 mM Na2HPO4 solutions containing 6-7 micrograms/mL protein, the apoC-1 monomer is approximately 30% alpha-helical at 0-22 degrees C and unfolds reversibly from about 22-80 degrees C with Tm = 51 +/- 3 degrees C and van't Hoff enthalpy delta Hv(Tm) = 19 +/- 3 kcal/mol. The apparent free energy of the monomer stabilization determined from the chemical unfolding at 0 degree C, delta G(0 degree C) = 2.8 +/- 0.8 kcal/mol, decreases by about 1 kcal/mol upon heating to 25 degrees C. A small apparent heat capacity increment suggests the absence of a substantial hydrophobic core for the apoC-1 molecule. At pH 7, increasing apoC-1 concentration above 10 micrograms/mL leads to self-association and formation of additional alpha-helices that unfold upon both heating and cooling from room temperature. The CD data indicate that the high-temperature transition reflects a complete monomer unfolding and the low-temperature transition reflects oligomer dissociation into stable monomers. This suggests the importance of hydrophobic interactions for apoC-1 self-association. Close proximity between the high- and low-temperature transitions and the absence of a plateau in the chemical unfolding curves recorded from oligomeric apoC-1 indicate marginal oligomer stability and suggest that in vivo apoC-1 transfer is mediated via the complexes with other apolipoproteins and/or lipids.     

Characterization of saxitoxin binding to saxiphilin, a relative of the transferrin family that displays pH-dependent ligand binding

Saxiphilin is a 91 kDa saxitoxin-binding protein that is homologous to members of the transferrin family of Fe(3+)-binding proteins noted for pH-dependent release of Fe3+. The mechanism of toxin binding to purified native saxiphilin from the bullfrog (Rana catesbeiana) was studied using [3H]saxitoxin. At pH 7.4 and 0 degrees C [3H]saxitoxin binds to a single site on saxiphilin with a KD of approximately 0.2 nM. The pH dependence of [3H]saxitoxin binding follows a one-site titration curve in the range of pH 9-4 with maximal binding from pH 9 to 7 and half-inhibition at pH 5.7. Inhibition of toxin binding at low pH is the combined result of a decrease in the rate of toxin association and an increase in the rate of toxin dissociation. The dependence of the apparent rate constants for [3H]saxitoxin association and dissociation on [H+] can be accounted for by a four-state model of allosteric interaction between the toxin-binding site and a single titratable residue of saxiphilin with a pKa of 7.2 in the toxin-free form and 4.3 in the toxin-bound form. From 0 to 25 degrees C, the temperature dependence of [3H]saxitoxin binding to saxiphilin is characterized by delta H degrees = -8.3 kcal mol-1, delta S degrees = 13.8 cal mol-1 K-1, and activation energies of 22.5 kcal mol-1 for dissociation and 11.1 kcal mol-1 for association. Binding of [3H]saxitoxin to saxiphilin is competitively inhibited with low affinity by a variety of divalent metal and lanthanide cations. Inhibition of toxin binding by the carboxyl-methylating reagent trimethyloxonium is prevented by pre-equilibration with [3H]saxitoxin, implicating the presence of one or more carboxyl groups in the binding site. Functional similarities suggest that the saxitoxin-binding site of saxiphilin is located in an interdomain cleft analogous to the location of one of the two homologous Fe(3+)-binding sites of transferrins. On the basis of residue substitutions between saxiphilin and transferrins, it is proposed that the saxitoxin-binding site is located in the carboxy terminal lobe of saxiphilin and that binding is modulated by protonation of a conserved histidine residue.     

Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.     

Design of a leucine zipper coiled coil stabilized 1.4 kcal mol-1 by phosphorylation of a serine in the e position

Using a dimeric bZIP protein, we have designed a leucine zipper that becomes more stable after a serine in the e position is phosphorylated by protein kinase A (delta delta GP = -1.4 kcal mol-1 dimer-1 or -0.7 kcal mol-1 residue-1). Mutagenesis studies indicate that three arginines form a network of inter-helical (i,i' + 5; i, i' + 2) and intra-helical (i, i + 4) attractive interactions with the phosphorylated serine. When the arginines are replaced with lysines, the stabilizing effect of serine phosphorylation is reduced (delta delta GP = -0.5 kcal mol-1 dimer-1). The hydrophobic interface of the leucine zipper needs a glycine in the d position to obtain an increase in stability after phosphorylation. The phosphorylated protein binds DNA with a 15-fold higher affinity. Using a transient transfection assay, we document a PKA dependent four-fold activation of a reporter gene. Phosphorylation of a threonine in the same e position decreases the stability by delta delta GP = +1.2 kcal mol-1 dimer-1. We present circular dichroism (CD) thermal denaturations of 15 bZIP proteins before and after phosphorylation. These data provide insights into the structural determinants that result in stabilization of a coiled coil by phosphorylation.     

Ultratight DNA binding of a new bisintercalating anthracycline antibiotic

Differential scanning calorimetry and absorption spectroscopy were used to characterize the interaction of the new bisintercalating anthracycline antibiotic, WP631, with DNA. The method of continuous variations revealed five distinct binding modes for WP631, corresponding to 6, 3, 1.3, 0.5, and 0.25 mol of base pairs (bp) per mole of ligand. The binding of one drug to 6 bp corresponds to the bisintercalative binding mode determined previously, and was the mode studied in detail. UV melting experiments and differential scanning calorimetry were used to measure the ultratight binding of WP631 to DNA. The binding constant for the interaction of WP631 with herring sperm DNA was determined to be 3.1 (+/- 0.2) x 10(11) M-1 at 20 degrees C. The large, favorable binding free energy of -15.3 kcal mol-1 was found to result from a large, negative enthalpic contribution of -30.2 kcal mol-1. DNA melting curves at different concentrations of WP631 were fitted to McGhee's model of DNA melting in the presence of ligands, yielding an independent estimate of DNA binding parameters. The salt dependence of the WP631 binding constant was examined, yielding a slope SK = delta (log K)/delta (log[Na+]) = 1.63. The observed salt dependence of the equilibrium constant, interpreted according to polyelectrolyte theory, indicates that there is a significant nonpolyelectrolyte contribution to the binding free energy. DNA melting studies using a homogeneous 214 bp DNA fragment showed that WP631 binds preferentially to the GC-rich region of the DNA.     

Recognition between disordered states: kinetics of the self-assembly of thioredoxin fragments

The disordered N- (1-73) and C- (74-108) fragments of oxidized Escherichia colithioredoxin (Trx) reconstitute the native structure upon association [Tasayco, M. L., & Chao, K. (1995) Proteins: Struct., Funct., Genet. 22, 41-44]. Kinetic measurements of the formation of the complex (1-73/74-108) at 20 degrees C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association [kon = (1330 +/- 54) M-1 s-1], and two apparent unimolecular rate constants [k1 = (0. 037 +/- 0.007) s-1 and k2 = (0.0020 +/- 0.0005) s-1]. The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants. An excess of N- over C-fragment decreases the kon, and the slowest phase disappears when a P76A variant is used. Stopped-flow fluorescence measurements at 20 degrees C indicate a GuHCl-dependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total. Their rate constants, extrapolated to zero denaturant, k-1 = (9 +/- 3) x 10(-5) s-1 and k-2 = (3.4 +/- 1.2) x 10(-5) s-1, show m# values of (4.0 +/- 0.4) kcal mol-1 M-1 and (3.5 +/- 0.1) kcal mol-1 M-1, respectively. Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment.     

Isotopic survival criteria of transfused polymorphonuclear cells

The enthalpies of interaction of urea with five globular proteins, ribonuclease A, trypsin, beta-lacto-globulin, ovalbumin and bovine serum albumin have been measured in aqueous solution at pH 7.0, I=0.005 M and 25 degrees C over a range of urea molality m from 0-15 mmol g-1 (where a 1 molal solution contains 1 mmol g-1). For all the proteins the interaction is exothermic, and there is an appreciable heat evolution at low urea concentrations, m less than 5 mmol g-1, which increases sharply at higher urea concentrations when the proteins undergo unfolding. If account is taken of the endothermic enthalpies of unfolding of the native proteins, the enthalpies of interactions of urea per unit mass denatured protein lie in the range -45 to -75 J g-1, corresponding to an average binding enthalpy of -23 kJ mol-1 bound urea.     

Conformation and unfolding thermodynamics of epidermal growth factor and derivatives

Mouse submaxillary epidermal growth factor (EGF) is a 53-residue single chain peptide hormone of known amino acid sequence which contains three disulfides, five tyrosines, and two tryptophans. Circular dichroic (CD) spectra have been obtained and resolved for EGF, several well-characterized chemical and enzymic derivatives, and related low molecular weight model compounds. Assignments have been made to most of the resolved bands; these include the peptide, aromatic, and disulfide chromophores. From a comparison of the rotational strength of the 213-nm resolved CD band in native EGF with that of standard proteins, EGF is estimated to contain about 22% beta structure and no alpha helicity. A derivative of EGF lacking the five carboxyl-terminal residues (prepared by limited trypsin digestion) and the cyanogen bromide derivative, in which there is a single main-chain cleavage at residue 21, have spectra properties indicative of approximately 10 and 12% beta structure, respectively. The near-ultraviolet CD spectra of the derivatives are similar to, albeit not identical with, that of EGF. The rotational strengths characteristic of the side-chain chromophores in EGF and these derivatives are several-fold higher than the corresponding values in low molecular weight model compounds. Thus, it appears that EGF and these modified forms contain a stable (and similar) tertiary structure. In contrast, the S-aminoethylated derivative of EGF exhibits a drastically altered CD spectrum relative to EGF indicating a different conformation(s). Equilibrium studies on the guanidinium hydrochloride (GdmCl) mediated reversible unfolding of EGF showed that the transition midpoint is quite high (i.e., 6.89 M GdmCl at 25.0 degrees C), thus, indicating considerable stability. From these data a rough estimate of 16 kcal/mol can be made for the unfolding free energy (delta G degrees) of EGF in the absence of denaturant. Interestingly, EGF exhibits greater stability characteristics than several proteins two to four times its size. The cyanogen bromide derivative of EGF exhibited greatly reduced stability characteristics, e.g., the transition midpoint occurred at 4.19 M GdmCl (25.0 degrees C) and delta G degrees was estimated to be approximately 4 kcal/mol. Thus, a single main-chain cleavage reduced the stability of EGF by about 70%. Thermal transitions of EGF and the cyanogen bromide derivative in the presence of concentrated GdmCl are characterized by a relatively high enthalpy of about 25 kcal/mol at 40 degrees C and a low (probably zero) heat capacity. From these thermodynamic parameters one can calculate that the large reduction in delta G degrees due to scission of the single peptide bond between residues 21 and 22 can be attributed almost completely to a change in entropy; e.g., at 40 degrees C the apparent entropy of unfolding of EGF is 20.4 cal mol-1 deg-1 while that of the cyanogen bromide derivative is 66.4 cal mol-1 deg-1.     

Urea and thermal equilibrium denaturation studies on the dimerization domain of Escherichia coli Trp repressor

The urea-induced equilibrium unfolding of the Escherichia coli Trp repressor (TR) is a two-state process, involving the native dimeric and unfolded monomeric species. Kinetic studies, however, reveal the presence of transient intermediates that appear only during the folding of the 107-residue protein [Gittelman, M. G., & Matthews, C. R. (1990) Biochemistry 29, 7011-7020]. In order to gain insight into the complex kinetic folding mechanism, the sequence of TR was reduced to the amino-terminal 66 residues, corresponding to the dimerization domain. Two polypeptides, 2-66 and NHis-7-66, were shown to be dimeric at 25 degrees C by size exclusion chromatography and to retain native-like spectroscopic features as evidenced by near- and far-UV circular dichroism and fluorescence spectroscopy. The equilibrium properties of the urea-induced folding of these core fragments were examined by intrinsic tryptophan fluorescence and circular dichroism and found to be well described by a two-state model. At 25 degrees C, the stabilities of both fragments are 14 kcal mol(-1), as compared to the 24 kcal mol(-1) observed for full-length TR. In contrast, the thermal denaturation of [2-66]2 and full-length TR are three-state processes; the midpoint of the transition monitored by absorbance at 292 nm precedes that monitored by circular dichroism at 222 nm. Global analysis of the thermal data as a function of monomer concentration suggests that both the full-length and [2-66]2 TR variants unfold via a dimeric intermediate. Taken together, these results demonstrate that the [2-66]2 fragment constitutes a well-structured, independently folding subdomain of TR that may be useful in elucidating the properties of the transient intermediates observed in the folding of the full-length protein. The dimeric intermediate observed in the thermal denaturation of [2-66]2 suggests that it may be possible to further reduce the core sequence while maintaining the ability to dimerize.     

Differential inhibition of lysophosphatidate signaling by volatile anesthetics

cAMP receptor protein (CRP) is involved in regulation of expression of several genes in Escherichia coli. The protein is a homodimer and each monomer is folded into two distinct structural domains. The mechanism of the biological activity of the protein may involve the interaction between the subunits and domains. In order to determine the interaction between the subunits or domains of CRP, we have studied the reversible denaturation of the protein by guanidine hydrochloride. The unfolding and refolding kinetics of CRP was monitored using stopped-flow fluorescence spectroscopy at 20 degrees C and pH 7.9. The results of CRP denaturation indicate that the transition can be described by a three-state model: (CRP native)2<=> 2 (CRP native)<=>2 (CRP denatured). The faster process, characterized by the relaxation time tau 2 = 80 +/- 3 ms, corresponds to the dissociation of CRP dimer into monomers. The slower process has the relaxation time tau t = 1.9 +/- 0.1 s and corresponds to the cooperative unfolding of CRP monomer. The free energy change in the absence of denaturant upon CRP dissociation is delta G dis degrees = 46.9 +/- 2.5 kJ/mol and for monomer unfolding delta G unf degrees = 30.9 +/- 1.3 kJ/mol. The thermal unfolding of CRP was studied by circular dichroism and fluorescence spectroscopy at various guanidine hydrochloride concentrations. It has been found that the native protein is maximally stable at about 21 +/- 0.3 degrees C and is denatured upon heating and cooling from this temperature. The apparent free energy change for CRP unfolding at 21 degrees C is equal to 30.5 +/- 0.4 kJ/mol and the apparent specific heat change is equal to delta Cp, app = 10.7 +/- 0.7 kJ mol-1 K-1. The predicted values of cold denaturation midpoint is equal to tau G = -18.8 +/- 1.5 degrees C and for high-temperature transition tau G = 63.1 +/- 1.5 degrees C. The predicted midpoint of high-temperature unfolding transition is about the same as determined experimentally.     

Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck

The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways. Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC). This is the first report of the use of ITC to study SH2 domain binding reactions. The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed. Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8). Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude. We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry. No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide. We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS)