Complexity in the redox titration of the dihaem cytochrome c4

Redox titration of the dihaem, two domain cytochromes c4 from Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii showed complex behaviour indicative of the presence of two redox components. In the case of the P. stutzeri cytochrome c4, two spectroscopically distinct components were present during the redox titration. In contrast, cytochrome c-554(548) from a halophilic Paracoccus species is a stable dimer of a monohaem cytochrome which shows close homology to cytochrome c4, but does not show complexity in its redox titration. The presence of chemically distinct haem environments or anti-cooperative interactions between identical haem groups are two possible explanations for the redox complexity of cytochrome c4. The simple redox titration of cytochrome c-554(548) shows that haems situated relatively close together need not interact, but direct cleavage, separation and study of the domains will be necessary to decide whether they do or do not interact in the case of cytochrome c4.     

Modelling of the human papillomavirus type 16 E5 protein

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7-9.9, at which 68-74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.     

Folding character of cytochrome c studied by o-nitrobenzyl modification of methionine 65 and subsequent ultraviolet light irradiation

The protein folding character of cyt c was studied with the use of a photocleavable o-nitrobenzyl derivative of Met65 (NBz-Met65). For the NBz-Met65 cyt c, the Soret absorption band slightly blue shifted compared with the unlabeled cyt c, the 695 nm absorption band related to the Met80 sulfur ligation to the heme iron disappeared, and its resonance Raman spectrum was characteristic of a six-coordinate low-spin species, all characters demonstrating coordination of a non-native ligand, probably a histidine, instead of Met80 to the heme iron. The far-UV circular dichroism (CD) spectrum of cyt c was altered, and the transition midpoint concentration value of guanidine hydrochloride (GdnHCl) for unfolding the protein decreased by 0.9 M by the modification, which showed perturbation of the structure and decrease in protein stability, respectively. With irradiation of 308 nm laser pulses on the NBz-Met65 cyt c, the Soret absorption band slightly red shifted, the 695 nm absorption band appeared, and the CD spectrum shifted toward that of the native protein, which demonstrated recovery of the methionine heme coordination and the native protein structure, due to reconversion of NBz-Met65 to unlabeled methionine. A fast phase was detected as a change in Soret absorbance with a rate constant of 21 000 +/- 4000 s(-)(1) during refolding of cyt c initiated by irradiation of a 308 nm pulse on the NBz-Met65 cyt c in the presence of 2 M GdnHCl. The observed rate constant corresponded well with that reported by the tryptophan fluorescence study [Shastry, M. C. R. S., and Roder, H. (1998) Nat. Struct. Biol. 5, 385-392]. The intermediate decayed with a rate constant of 90 +/- 15, followed by another phase with a rate constant of 13 +/- 3 s(-)(1), and was not seen in the absence of GdnHCl.     

Structural transformation of cytochrome c and apo cytochrome c induced by sulfonated polystyrene

The structural transformation of cytochrome c (cyt c) and its heme-free precursor, apo cyt c, induced by negatively charged sulfonated polystyrene (SPS) with different charge density (degree of sulfonation) and chain length was studied to understand the factors that influence the folding and unfolding of the protein. SPS forms stable transparent nanoparticles in aqueous solution. The hydrophobic association of the backbone chain and phenyl groups is balanced by the electrostatic repulsion of the sulfonate groups on the particle surface. The binding of cyt c to negatively charged SPS particles causes an extensive disruption of the native compact structure of cyt c: the cleavage of Fe-Met80 ligand, about 40% loss of the helical structure, and the disruption of the asymmetry environment of Trp59. On the other hand, SPS particle-bound apo cyt c undergoes a conformational change from the random coil to alpha-helical structure. The folding of apo cyt c in SPS particles was influenced by pH and ionic strength of the solution, SPS concentration, and the degree of sulfonation and chain length of SPS. The folding can reach more than 90% of the alpha-helix content of native cyt c in solution. Poly(sodium 4-styrenesulfonate) (PSS), which is 100% sulfonated polystyrene and cannot form hydrophobic cores in the solution, induces only two-thirds of the alpha-helix content compared with SPS. It appears that the electrostatic interaction between PSS/SPS and apo cyt c induces an early partially folded state of apo cyt c. The hydrophobic interaction between nonpolar residues in apo cyt c and the hydrophobic cores in SPS particles extends the alpha-helical structure of apo cyt c.     

Folding of horse cytochrome c in the reduced state

Equilibrium and kinetic folding studies of horse cytochrome c in the reduced state have been carried out under strictly anaerobic conditions at neutral pH, 10 degrees C, in the entire range of aqueous solubility of guanidinium hydrochloride (GdnHCl). Equilibrium unfolding transitions observed by Soret heme absorbance, excitation energy transfer from the lone tryptophan residue to the ferrous heme, and far-UV circular dichroism (CD) are all biphasic and superimposable, implying no accumulation of structural intermediates. The thermodynamic parameters obtained by two-state analysis of these transitions yielded DeltaG(H2O)=18.8(+/-1.45) kcal mol(-1), and C(m)=5.1(+/-0.15) M GdnHCl, indicating unusual stability of reduced cytochrome c. These results have been used in conjunction with the redox potential of native cytochrome c and the known stability of oxidized cytochrome c to estimate a value of -164 mV as the redox potential of the unfolded protein. Stopped-flow kinetics of folding and unfolding have been recorded by Soret heme absorbance, and tryptophan fluorescence as observables. The refolding kinetics are monophasic in the transition region, but become biphasic as moderate to strongly native-like conditions are approached. There also is a burst folding reaction unobservable in the stopped-flow time window. Analyses of the two observable rates and their amplitudes indicate that the faster of the two rates corresponds to apparent two-state folding (U<-->N) of 80-90 % of unfolded molecules with a time constant in the range 190-550 micros estimated by linear extrapolation and model calculations. The remaining 10-20 % of the population folds to an off-pathway intermediate, I, which is required to unfold first to the initial unfolded state, U, in order to refold correctly to the native state, N (I<-->U<-->N). The slower of the two observable rates, which has a positive slope in the linear functional dependence on the denaturant concentration indicating that an unfolding process under native-like conditions indeed exists, originates from the unfolding of I to U, which rate-limits the overall folding of these 10-20 % of molecules. Both fast and slow rates are independent of protein concentration and pH of the refolding milieu, suggesting that the off-pathway intermediate is not a protein aggregate or trapped by heme misligation. The nature or type of unfolded-state heme ligation does not interfere with refolding. Equilibrium pH titration of the unfolded state yielded coupled ionization of the two non-native histidine ligands, H26 and H33, with a pK(a) value of 5.85. A substantial fraction of the unfolded population persists as the six-coordinate form even at low pH, suggesting ligation of the two methionine residues, M65 and M80. These results have been used along with the known ligand-binding properties of unfolded cytochrome c to propose a model for heme ligation dynamics. In contrast to refolding kinetics, the unfolding kinetics of reduced cytochrome c recorded by observation of Soret absorbance and tryptophan fluorescence are all slow, simple, and single-exponential. In the presence of 6.8 M GdnHCl, the unfolding time constant is approximately 300(+/-125) ms. There is no burst unfolding reaction. Simulations of the observed folding-unfolding kinetics by numerical solutions of the rate equations corresponding to the three-state I<-->U<-->N scheme have yielded the microscopic rate constants.     

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

The bacterial cytochrome c peroxidases contain an electron-transferring haem c (E) and a peroxidatic haem c (P). Many are isolated in an inactive oxidised state. Reduction of the E haem promotes Ca(2+)-dependent spin state and coordination changes at the P haem rendering it accessible to ligand. Recent crystallographic work on the oxidised and mixed valence enzymes has suggested a mechanism by which an electron entering the E haem remotely triggers this activation of the P haem. Binding of hydrogen peroxide at the activated P haem leads to an intermediate catalytic form containing two oxidising equivalents, one of which is a ferryl oxene. This form of the enzyme is then reduced by two single electron transfers to the E haem delivered by small redox proteins such as cytochromes or cupredoxins. The binding of these small redox proteins is dominated by global electrostatic forces but the interfaces of the electron transfer complexes that are formed are largely hydrophobic and relatively non-specific. These features allow very high electron transfer rates in the steady state.     

Two-dimensional infrared correlation spectroscopy as a probe of sequential events in the thermal unfolding of cytochromes c.

The sequential unfolding events of horse, cow, and tuna ferricytochromes c (cyt c) as a function of increasing temperature over the range 25-81 degrees C were investigated by resolution-enhanced two-dimensional infrared (2D IR) correlation spectroscopy. The 2D IR analysis revealed that in the thermal denaturation of the two mammalian cyts, the overall sequence of unfolding is similar, with denaturation of extended-chain and turn structures occurring prior to unfolding of alpha-helices, followed by denaturation of residual stable extended-chain structures. In tuna cyt c, denaturation of all extended-chain structures precedes the unfolding of alpha-helices. Moreover, in cow cyt c, unfolding of all helical components occurs as one cooperative unit, but in horse and tuna cyts c, the helical components behave as subdomains that unfold separately, as proposed recently by Englander and co-workers for horse cyt c [Bai et al. (1995) Science 269, 192-197; Milne et al. (1999) J. Mol. Biol. 290, 811-822]. At higher temperatures, following the loss of secondary structure, protein aggregation occurs in the three cyts c. The data presented here establish that variations in the thermal unfolding of cyts c can be associated with specific sites in the protein that influence local flexibility yet have little affect on global stability. This study demonstrates the power of resolution-enhanced 2D IR correlation spectroscopy in probing unfolding events in homologous proteins.     

The folding mechanism of a two-domain protein: folding kinetics and domain docking of the gene-3 protein of phage fd

The gene-3 protein (G3P) of filamentous phages is essential for the infection of Escherichia coli. The carboxy-terminal domain anchors this protein in the phage coat, whereas the two amino-terminal domains N1 and N2 protrude from the phage surface. We analyzed the folding mechanism of the two-domain fragment N1-N2 of G3P (G3P(*)) and the interplay between folding and domain assembly. For this analysis, a variant of G3P(*) was used that contained four stabilizing mutations (IIHY-G3P(*)). The observed refolding kinetics extend from 10 ms to several hours. Domain N1 refolds very rapidly (with a time constant of 9.4 ms at 0.5 M guanidinium chloride, 25 degrees C) both as a part of IIHY-G3P(*) and as an isolated protein fragment. The refolding of domain N2 is slower and involves two reactions with time constants of seven seconds and 42 seconds. These folding reactions of the individual domains are followed by a very slow, spectroscopically silent docking process, which shows a time constant of 6200 seconds. This reaction was detected by a kinetic unfolding assay for native molecules. Before docking, N1 and N2 unfold fast and independently, after docking they unfold slowly in a correlated fashion. A high energy barrier is thus created by domain docking, which protects G3P kinetically against unfolding. The slow domain docking is possibly important for the infection of E.coli by the phage. Upon binding to the F pilus, the N2 domain separates from N1 and the binding site for TolA on domain N1 is exposed. Since domain reassembly is so slow, this binding site remains accessible until pilus retraction has brought N1 close to TolA on the bacterial surface.     

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.     

Unfolding transitions of fibronectin and its domains. Stabilization and structural alteration of the N-terminal domain by heparin.

Changes in the conformational state of human plasma fibronectin and several of its fragments were studied by fluorescence emission, intrinsic fluorescence polarization and c.d. spectroscopy under conditions of guanidinium chloride-and temperature-induced unfolding. Fragments were chosen to represent all three types of internal structural homology in the protein. Low concentration (less than 2 M) of guanidinium chloride induced a gradual transition in the intact protein that was not characteristic of any of the isolated domains, suggesting the presence of interdomain interactions within the protein. Intermediate concentrations of guanidinium chloride (2-3 M) and moderately elevated temperatures (55-60 degrees C) induced a highly co-operative structural transition in intact fibronectin that was attributable to the central 110 kDa cell-binding domain. High temperatures (greater than 60 degrees C) produced a gradual unfolding in the intact protein attributable to the 29 kDa N-terminal heparin-binding and 40 kDa collagen-binding domains. Binding of heparin to intact fibronectin and to its N-terminal fragment stabilized the proteins against thermal unfolding. This was reflected in increased delta H for the unfolding transitions of the heparin-bound N-terminal fragment, as well as decreased accessibility to solvent perturbants of internal chromophores in this fragment when bound to heparin. These results help to account for the biological efficacy of the interaction between the fibronectin N-terminal domain and heparin, despite its relatively low affinity.     

Thermodynamic study of phosphoglycerate kinase from Thermotoga maritima and its isolated domains: reversible thermal unfolding monitored by differential scanning calorimetry and circular dichroism spectroscopy

The folding of phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima and its isolated N- and C-terminal domains (N1/2 and C1/2) was characterized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. At pH 3.0-4.0, reversible thermal denaturation of TmPGK occurred below 90 degrees C. The corresponding peaks in the partial molar heat capacity function were fitted by a four-state model, describing three well-defined unfolding transitions. Using CD spectroscopy, these are ascribed to the disruption of the domain interactions and subsequent sequential unfolding of the two domains. The isolated N-terminal domain unfolds reversibly between pH 3.0 and pH 4.0 to >90% and at pH 7.0 to about 70%. In contrast, the isolated engineered C-terminal domain only shows reversible thermal denaturation between pH 3.0 and pH 3.5. Neither N1/2 nor C1/2 obeys the simple two-state mechanism of unfolding. Instead, both unfold via a partially structured intermediate. In the case of N1/2, the intermediate exhibits native secondary structure and perturbed tertiary structure, whereas for C1/2 the intermediate could not be defined with certainty.     

Denaturation behavior of antithrombin in guanidinium chloride. Irreversibility of unfolding caused by aggregation

The structural stability of the protease inhibitor antithrombin from bovine plasma was examined as a function of the concentration of guanidinium chloride (GdmCl). A biphasic unfolding curve at pH 7.4, with midpoints for the two phases at 0.8 and 2.8 M GdmCl, was measured by far-ultraviolet circular dichroism. Spectroscopic and hydrodynamic analyses suggest that the intermediate state which exists at 1.5 M GdmCl involves a partial unfolding of the antithrombin molecule that exposes regions of the polypeptide chain through which slow, intermolecular association subsequently takes place. The partially unfolded molecule can be reversed to its fully functional state only before the aggregation occurs. Upon return of the aggregated state to dilute buffer, the partially unfolded antithrombin remains aggregated and does not regain the spectroscopic properties, thrombin-inhibitory activity, or heparin affinity of the native inhibitor. This behavior indicates that the loss of the functional properties of the proteins is caused by the macromolecular association. Comparative experiments gave similar results for the human inhibitor. Analyses of bovine antithrombin in 6 M GdmCl indicated that the second transition reflects the total unfolding of the protein to a disulfide-cross-linked random coil. This transition is spectroscopically reversible; however, on further reversal to dilute buffer, the molecules apparently are trapped in the partially unfolded, aggregated, intermediate state. The results are consistent with the existence of two separate domains in antithrombin which unfold at different concentrations of GdmCl but do not support the contention that the thrombin-binding and heparin-binding regions of the protein are located in different domains [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053].     

How does reorganization energy change upon protein unfolding? Monitoring the structural perturbations in the heme cavity of cytochrome c

In several classes of proteins the redox center provides an additional intrinsic biophysical probe that could be used to study the protein structure and function. In present report reorganization energy (lambda, as a parameter describing electron transfer properties) was used to study the protein structural changes around the heme prosthetic group in cytochrome c (cyt c). We attempted to monitor the value of this parameter upon the unfolding process of cyt c by urea, during which it was increased sigmoidally from about 0.52 to 0.82 eV for native and unfold protein, respectively. Results indicate that by structural changes in the heme site, lambda provides a complementary tool for following the unfolding process. Assuming a reversible two-state model for cyt c unfolding, Delta G(H2O), Cm and m values were determined to be 8.32+/-0.7 kcal mol(-1), 1.53+/-0.19 kcalmol(-1)M(-1) and 5.03 M, respectively.     

Nature of the fast and slow refolding reactions of iron(III) cytochrome c

The fast and slow refolding reactions of iron(III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. (Ikai, A., Fish, W. W., & Tanford, C. (1973) J. Mol. Biol. 73, 165--184), have been reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25 degrees C, 1.75 M guanidine hydrochloride). We show here that native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes have been used to test for formation of native cyt c. absorbance in the 695-nm band and rate of reduction of by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown both by refolding assays at different times after unfolding ("double-jump" experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF leads to N and the slow refolding reaction is Us leads to N, where N is native cyt c, and there is a US in equilibrium UF equilibrium in unfolded cyt c. The results are consistent with the UF in equilibrium US reaction being proline isomerization, but this has not yet been tested in detail. Folding intermediates have been detected in both reactions. In the UF leads to N reaction, the Soret absorbance change precedes the recovery of the native 695-nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US leads to N reaction an ascorbate-reducible intermediate has been found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.     

Unfolding the A2 Domain of Von Willebrand Factor with the Optical Trap

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein involved in both hemostasis and thrombosis. VWF conformational changes, especially unfolding of the A2 domain, may be required for efficient enzymatic cleavage in vivo. It has been shown that a single A2 domain unfolds at most probable unfolding forces of 7-14 pN at force loading rates of 0.35-350 pN/s and A2 unfolding facilitates A2 cleavage in vitro. However, it remains unknown how much force is required to unfold the A2 domain in the context of a VWF multimer where A2 may be stabilized by other domains like A1 and A3. With the optical trap, we stretched VWF multimers and a poly-protein (A1A2A3)₃ that contains three repeats of the triplet A1A2A3 domains at constant speeds of 2000 nm/s and 400 nm/s, respectively, which yielded corresponding average force loading rates of 90 and 22 pN/s. We found that VWF multimers became stiffer when they were stretched and extended by force. After force increased to a certain level, sudden extensional jumps that signify domain unfolding were often observed. Histograms of the unfolding force and the unfolded contour length showed two or three peaks that were integral multiples of ∼21 pN and ∼63 nm, respectively. Stretching of (A1A2A3)₃ yielded comparable distributions of unfolding force and unfolded contour length, showing that unfolding of the A2 domain accounts for the behavior of VWF multimers under tension. These results show that the A2 domain can be indeed unfolded in the presence of A1, A3, and other domains. Compared with the value in the literature, the larger most probable unfolding force measured in this study suggests that the A2 domain is mechanically stabilized by A1 or A3 although variations in experimental setups and conditions may complicate this interpretation.     

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.     

ATP specifically drives refolding of non-native conformations of cytochrome c

An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.     

Folding of the multidomain ribosomal protein L9: the two domains fold independently with remarkably different rates

The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (Tm = 67 degrees C for the C-terminal domain, 80 degrees C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72 degrees C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69 degrees C were used to determine the folding rate constant, 3.3 s-1, and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1, respectively, for the N-terminal domain at 25 degrees C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N- and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.     

Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.     

Probing the folded state of fibronectin type III domains in stretched fibrils by measuring buried cysteine accessibility

Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.