Immunochemical determination of conformational equilibria for fragments of the B beta chain of fibrinogen

The conformations of the B beta chain of the intact fibrinogen molecule and of various fragments of the B beta chain of fibrinogen that contain the region that is hydrolyzed by thrombin have been compared by an immunochemical method [Sachs, D. H., Schechter, A. N., Eastlake, A., & Anfinsen, C. B. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3790]. Anti-fibrinogen antibodies were induced in rabbits by immunization with native bovine fibrinogen. An antibody population specific for the native antigenic determinant within the B beta fragment 20-28 was isolated by immunoadsorption. This preparation was to determine the value of Kconf, the equilibrium constant for the interconversion of the nonnative and native conformations of this determinant. Values of Kconf were measured for this determinant within native fibrinogen, the disulfide knot (DSK), CNBrB beta, B beta fragment 16-28, B beta fragment 20-28, and fibrinopeptide B (FpB). 125I-Labeled fibrinogen (125I-F) was used in the determination of Kconf by measuring the competition between 125I-F and the fibrinogen derivatives under study for binding to the purified antibody. For the antigenic region in F, the DSK, and CNBrB beta, the values of Kconf at 4 degrees C were infinity, (5.9 +/- 3.5) X 10(-3), and (1.2 +/- 0.7) X 10(-3), respectively. The values of Kconf for B beta fragment 16-28, B beta fragment 20-28, and FpB at 4 degrees C were less than (6.0 +/- 3.9) X 10(-7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional effects of heme orientational disorder in sperm whale myoglobin

The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically at pH 8, 20 degrees C. The conventional absorbance and magnetic circular dichroism spectra of freshly reconstituted samples were identical to those of the native protein. In contrast, reconstituted azide or CO myoglobin initially exhibited less circular dichroism in the Soret wavelength region than native myoglobin. These data support the theory proposed by La Mar and co-workers (La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, R. M. (1983) J. Mol. Biol. 168, 887-896) that protoheme inserts into apomyoglobin in two distinct orientations. The equilibrium and kinetic parameters for O2 and CO binding to newly reconstituted myoglobin were observed to be identical to those of the native protein. Thus, the orientation of the heme group has no effect on the physiological properties of myoglobin. This result is in disagreement with the preliminary report of Livingston et al. (Livingston, D. J., Davis, N. L., La Mar, G. N., and Brown, W. D. (1984) J. Am. Chem. Soc. 106, 3025-3026) which suggested that the abnormal heme conformation exhibited a 10-fold greater affinity and association rate constant for O2 binding. Significant kinetic heterogeneity was observed only for long-chain isonitrile binding to newly reconstituted myoglobin, and even in these cases, the rate constants for the abnormal and normal heme conformations differed by less than a factor of 4.     

Quenching of the zinc-protoporphyrin triplet state as a measure of small-molecule diffusion through the structure of myoglobin

The diffusion of small molecules through the myoglobin structure was studied. It has been shown that the fluorescent Zn-protoporphyrin substitutes easily for the native nonfluorescent Fe-protoporphyrin in myoglobin. The quenching rate of the E-type delayed fluorescence of Zn-protoporphyrin in a substituted myoglobin by the quenchers oxygen and anthraquinonesulfonate was used to measure their diffusion from the ambient solution through the protein to the ligand binding site. The quenching rate constant (at 21 degrees C) for oxygen is kq = (9.6 +/- 0.9) X 10(7) M-1 S-1, only 1 order of magnitude less than that for Zn-hematoporphyrin quenching in aqueous solution. The activation energy in the range between 2 and 40 degrees C is Ea = 6.0 +/- 0.6 kcal/mol. The corresponding data for anthraquinonesulfonate are kq = (2.1 +/- 0.3) X 10(8) M-1 S-1 and Ea = 5.8 +/- 0.6 kcal/mol. Taking into account the statistical factor involved in the oxygen quenching of the Zn-porphyrin triplet, the quenching rates are very similar. The data are discussed in terms of the "gated reaction" theory of Northrup and McCammon. The similar rate constants and activation energies indicate that the diffusion rate in the protein is determined by the frequency of the conformational changes that open "gates" for the passage of the quencher through the protein.     

Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.     

NMR detection of multiple transitions to low-populated states in azurin

Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone (15)N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8 degrees C). Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes. The group of (15)N nuclei involved in the faster process exhibits at 44.8 degrees C a forward rate constant of 11.7+/-2.4 s(-1) and a population of the exited state of 0.39+/-0.07%. They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane. For the slower process, the forward rate constant and population of the exited state at 44.8 degrees C are 4.1+/-0.1 s(-1) and 0.45+/-0.02%, respectively. The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 A, indicating conformational rearrangements involving the copper coordinating loops. The dependence of the equilibrium constant on the temperature is consistent with an enthalpy-dominated transition around the copper, but an entropy-controlled transition near histidine 35. The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy-cost rearrangement of the copper-binding site that may be necessary for efficient electron transfer.     

Influence of hemin on the conformation of cyanogen bromide-cleaved peptides of apomyoglobin

The complexes of the three BrCN-cleaved fragments of sperm whale apomyoglobin with hemin were studied by circular dichroism (CD). In native myoglobin, the heme is located in the middle fragment; the isolated peptide (residues 56–131), however, produces little extrinsic Cotton effects by the addition of hemin, although about four molecules of hemin are bound to this peptide. In marked contrast, the COOH-terminal peptide (residues 132–153), which binds three hemin molecules, shows strong Cotton effects in the Soret bands and drastically changes its conformation from unordered to highly helical. The Arg-modified or Lys-deaminated peptide no longer undergoes conformational changes by the addition of hemin, suggesting that the two propionic acid groups of one hemin molecule interact with the Arg residue and one of the Lys residues, which stabilizes the induced helical conformation. The NH₂-terminal peptide (residues 1–55) binds one hemin molecules, and the helicity of this fragment is slightly enhanced by the addition of hemin. Both the CD and difference absorption spectra indicate that the mode of interaction between the peptides and hemin are different for the three apomyoglobin fragments.     

Conformation of a T cell stimulating peptide in aqueous solution

Using two-dimensional NMR spectroscopy and circular dichroism spectroscopy it is demonstrated that a T cell stimulating peptide corresponding to residues 132-153 of sperm whale myoglobin populates helical conformations in aqueous solution. This finding is in accordance with proposals that immunodominant sites in T cell stimulating peptides have a high conformational propensity. The observation of secondary structure in aqueous solutions of this and other immunogenic peptides has important implications for initiation of protein folding.     

Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI)

A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI). Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli. The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms. Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra. Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix. Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3. Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den). Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies. To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously. Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants. The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions. It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude. Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis.     

An isolated helix persists in a sparsely populated form of KIX under native conditions

NMR relaxation dispersion techniques were used to investigate conformational exchange of the three-helix bundle protein KIX under native conditions. These experiments provide site-resolved kinetic information about microsecond-to-millisecond time scale motions along with structural (chemical shift) information without requiring a perturbation of the equilibrium. All kinetic data are consistent with an apparent two-state transition between natively folded KIX and a partially unfolded high-energy state that is populated to 3.0 +/- 0.2% at 27 degrees C. By combining (13)C- and (15)N-based experiments that probe specific structural aspects, we show that the sparsely populated high-energy state displays a strong conformational preference. An isolated secondary structural element, C-terminal helix alpha3, is highly populated, while the hydrophobic core of the domain and the remainder of the protein backbone, including helices alpha1 and alpha2, are disordered and devoid of specific interactions. This high-energy state presumably represents the equilibrium analogue of a folding intermediate that is transiently populated in stopped-flow kinetic experiments [Horng, J. C., Tracz, S. M., Lumb, K. J., and Raleigh, D. P. (2002) Biochemistry 44, 627-634].     

Refolding of denatured ribonuclease observed by size exclusion chromatography

The unfolding and refolding of pancreatic ribonuclease have been observed by absorbance, fluorescence, and size exclusion chromatographic measurements in solutions of guanidinium chloride continuously maintained at pH 6.0 and 4 degrees C. The spectral measurements were fitted with a minimal number of kinetic phases while the chromatographic measurements were simulated from an explicit mechanism. All of the measurements are consistent with a minimal mechanism involving seven components. The folded components include the native protein and two transiently stable intermediates each having the same hydrodynamic volume. The intermediate having all native peptide isomers has an unfolding midpoint in 3.8 M denaturant while the intermediate having one nonnative peptide isomer has an unfolding midpoint in 1.3 M denaturant. The unfolded protein is distributed among four components having the same hydrodynamic volume but differing peptide isomers. At equilibrium, 10% of the denatured protein has all native isomers, 60% has one nonnative isomer, 5% has a different nonnative isomer, and 25% has both nonnative isomers. In low denaturant concentrations, the dominant component with one nonnative isomer can refold to transiently populate the compact intermediate with the same nonnative isomer.     

Localization of the metal-induced conformational transition of bovine prothrombin

The calcium-stabilized antigenic determinants on bovine prothrombin were localized to the NH2-terminal 1-42 residues using conformation-specific antibodies. Polyclonal antibodies to the bovine prothrombin-Ca(II) complex were raised in rabbits, and purified antibody subpopulations were isolated by sequential immunoabsorption and affinity chromatography. Anti-prothrombin-Ca(II) antibodies, characterized by their absolute specificity for the prothrombin-metal complex (Tai, M. M., Furie, B. C., and Furie, B. (1980) J. Biol. Chem. 255, 2790-2795), bound to prothrombin, fragment 1, reduced and carboxymethylated fragment 1, and CNBr fragment (1-72) in solution. However, these antibodies do not bind significantly to the gamma-carboxyglutamic acid-rich fragment (1-39), CNBr fragment (73-156), or prethrombin 1. To obviate the complex analysis of possible reasons for the lack of antibody binding to small peptides in solution, conformation-specific antibodies directed against defined regions of the whole prothrombin molecule were isolated. The influence of calcium ions on the binding of these site-specific antibody subpopulations to 125I-labeled prothrombin fragment 1 was evaluated. Anti-(1-39)N, anti-(1-42)N, anti-(1-72)N, and anti-(reduced and carboxymethylated fragment 1)N showed enhanced binding to prothrombin fragment 1 in the presence of Ca(II), indicating the presence of calcium-stabilized antigenic determinants within each of these regions on fragment 1. In contrast, calcium ions had no effect on the interaction of anti-des-(1-42)prothrombin, anti-prethrombin 1, anti-(43-72)N, and anti-(73-156)N antibodies with prothrombin fragment 1. These results indicate that the metal-induced conformational transition, monitored immunochemically, is localized to the NH2-terminal, gamma-carboxyglutamic acid-rich region of prothrombin between residues 1-42.     

Thermodynamic study of the binding of the tandem-SH2 domain of the Syk kinase to a dually phosphorylated ITAM peptide: evidence for two conformers

The cytosolic tyrosine kinase Syk is recruited to immune cell receptors via interactions of its tandem-SH2 domain with tyrosine-phosphorylated sequences called immune receptor tyrosine activation motifs (ITAMs). We have characterized the binding of the tandem-SH2 domain of Syk (Syk-tSH2) to a dually phosphorylated peptide derived from the ITAM of the T cell receptor CD3-epsilon subunit. The CD3-epsilon peptide binds with an affinity of 18-81 nM at 150 mM NaCl over the 4.5-30 degrees C temperature range that was studied. The enthalpy of binding, DeltaH degrees obs, shows an unusual nonlinear dependence on temperature, suggesting the possibility of a temperature-dependent conformational equilibrium coupled to binding. This hypothesis was tested and confirmed by examining the temperature dependence of (1) the on-rate constant for binding and (2) the fluorescence of Syk-tSH2 and its CD3-epsilon peptide complex. The DeltaH degrees obs, Kobs, fluorescence, and kinetic data are all well described by a model incorporating the hypothesized conformational equilibrium. Circular dichroism spectra at various temperatures indicate that the conformational change is not due to a partial unfolding of the protein. We suggest that the conformational equilibrium enables Syk-tSH2 to exhibit flexibility in its binding modality, which may be necessitated by Syk's involvement in a wide variety of signal tranduction pathways.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Folding thermodynamics and kinetics of YNMG RNA hairpins: specific incorporation of 8-bromoguanosine leads to stabilization by enhancement of the folding rate

Modified nucleotides allow fundamental energetic and kinetic properties of nucleic acids to be probed. Here, we demonstrate that an RNA hairpin containing the nucleotide analogue 8-bromoguanosine (8BrG or G), gcUUCGgc, has enhanced stability relative to the unmodified hairpin, with DeltaDeltaG(37)(degrees)= -0.69 +/- 0.15 kcal mol(-1) and DeltaT(M) = +6.8 +/- 1.4 degrees C. NMR spectroscopic data suggest that the enhanced stability of gcUUCGgc does not arise from the native state; laser temperature-jump experiments support this notion, as gcUUCGgc and gcUUCGgc have similar unfolding rate constants, but the folding rate constant of gcUUCGgc is 4.1-fold faster at 37.5 degrees C and 2.8-fold faster under isoenergetic conditions. On the basis of these findings, we propose that 8BrG reduces the conformational entropy of the denatured state, resulting in an accelerated conformational search for the native state and enhanced stability.     

Detection and characterization of two ATP-dependent conformational changes in proteolytically inactive Escherichia coli Lon mutants by stopped flow kinetic techniques

Lon is an ATP dependent serine protease responsible for degrading denatured, oxidatively damaged and certain regulatory proteins in the cell. In this study we exploited the fluorescence properties of a dansylated peptide substrate (S4) and the intrinsic Trp residues in Lon to monitor peptide interacting with the enzyme. We generated two proteolytically inactive Lon mutants, S679A and S679W, where the active site serine is mutated to an Ala and Trp residue, respectively. Stopped-flow fluorescence spectroscopy was used to identify key enzyme intermediates generated along the reaction pathway prior to peptide hydrolysis. A two-step peptide binding event is detected in both mutants, where a conformational change occurs after a rapid equilibrium peptide binding step. The Kd for the initial peptide binding step determined by kinetic and equilibrium binding techniques is approximately 164 micromolar and 38 micromolar, respectively. The rate constants for the conformational change detected in the S679A and S679W Lon mutants are 0.74 +/- 0.10 s(-1) and 0.57 +/- 0.10 s(-1), respectively. These values are comparable to the lag rate constant determined for peptide hydrolysis (klag approximately 1 s(-1)) [Vineyard, D., et al. (2005) Biochemistry 45, 4602-4610]. Replacement of the active site Ser with Trp (S679W) allows for the detection of an ATP-dependent conformational change within the proteolytic site. The rate constant for this conformational change is 7.6 +/- 1.0 s(-1), and is essentially identical to the burst rate constant determined for ATP hydrolysis under comparable reaction conditions. Collectively, these kinetic data support a mechanism by which the binding of ATP to an allosteric site on Lon activates the proteolytic site. In this model, the energy derived from the binding of ATP minimally supports peptide cleavage by allowing peptide substrate access to the proteolytic site. However, the kinetics of peptide cleavage are enhanced by the hydrolysis of ATP.     

Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity.     

Truncation analysis of several DR binding epitopes.

Peptide regions crucial for binding to four different DR alleles (DR1, DR2, DR5, and DR52a) have been localized in five unrelated DR binding peptides (dynorphin 1-13, sperm whale myoglobin 132-153, influenza hemagglutinin 307-319, pigeon cytochrome c 88-104, and tetanus toxoid 830-843) by testing panels of truncated analogs for DR binding. It was found that in most cases, different DR alleles recognize almost identical, albeit distinct, core regions, suggesting that different DR alleles may recognize similar structures on their peptide ligands. Furthermore, it was found that these core regions, notwithstanding their derivation from unrelated sequences, share a common structural pattern. When the sequences of several other unrelated determinants were scrutinized, the structural motif identified was present in some, but absent in other good DR binders, suggesting that good DR binding capacity of peptide molecules may be compatible with more than one single sequence pattern.     

Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: energetics of peptide association and folding

Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (Kassn approximately 1.4 x 10(5) M(-1) at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a DeltaC(p) degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn.HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Halpha chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.