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

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

Flavodoxin-cytochrome c interactions: circular dichroism and nuclear magnetic resonance studies

Circular dichroism and 1H and 31P nuclear magnetic resonance spectroscopy have been used to investigate complex formation between cytochrome c and the flavodoxins from Azotobacter vinelandii and Clostridium pasteurianum. Such complexes are known to be involved in the mechanism of electron transfer between these two redox proteins. A large increase in ellipticity in the Soret band of the cytochrome heme was observed upon formation of the Clostridium flavodoxin complex, whereas much smaller changes were found for the complexes with either Azotobacter flavodoxin or an 8 alpha-imidazolyl-FMN-substituted Clostridium flavodoxin analogue. Similarly, the magnitudes of the perturbations of the contact-shifted heme proton resonances obtained upon complexation of cytochrome c by Azotobacter flavodoxin were much smaller than those previously shown for Clostridium flavodoxin [Hazzard, J. T., & Tollin, G. (1985) Biochem. Biophys. Res. Commun. 130, 1281-1286]. 31P nuclear magnetic resonance measurements were also consistent with differences in the interactions between the components in the complexes of the two flavodoxins with cytochrome c. It is suggested that these spectral changes are due to a loosening or opening of the heme crevice upon Clostridium flavodoxin binding, which allows closer contact between the heme and flavin prosthetic groups and results in a faster rate of electron transfer. The implications of these observations for biological oxidation-reduction processes are considered.     

The structure of Escherichia coli heat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism.

The heat-stable enterotoxin b (STb) is secreted by enterotoxigenic Escherichia coli that cause secretory diarrhea in animals and humans. It is a 48-amino acid peptide containing two disulfide bridges, between residues 10 and 48 and 21 and 36, which are crucial for its biological activity. Here, we report the solution structure of STb determined by two- and three-dimensional NMR methods. Approximate interproton distances derived from NOE data were used to construct structures of STb using distance-geometry and simulated annealing procedures. The NMR-derived structure shows that STb is helical between residues 10 and 22 and residues 38 and 44. The helical structure in the region 10-22 is amphipathic and exposes several polar residues to the solvent, some of which have been shown to be important in determining the toxicity of STb. The hydrophobic residues on the opposite face of this helix make contacts with the hydrophobic residues of the C-terminal helix. The loop region between residues 21 and 36 has another cluster of hydrophobic residues and exposes Arg 29 and Asp 30, which have been shown to be important for intestinal secretory activity. CD studies show that reduction of disulfide bridges results in a dramatic loss of structure, which correlates with loss of function. Reduced STb adopts a predominantly random-coil conformation. Chromatographic measurements of concentrations of native, fully reduced, and single-disulfide species in equilibrium mixtures of STb in redox buffers indicate that the formation of the two disulfide bonds in STb is only moderately cooperative. Similar measurements in the presence of 8 M urea suggest that the native secondary structure significantly stabilizes the disulfide bonds.     

Studies of synthetic helical peptides using circular dichroism and nuclear magnetic resonance

We have designed a set of 17-residue synthetic peptides to be monomeric helices in aqueous solution. Circular dichrosim experiments indicate the presence of helical structure in aqueous solution at low temperature and low pH. The two-dimensional nuclear magnetic resonance results for one of the peptides show a segment of ten residues which clearly meets all of the criteria for the existence of helical structure at both 5 degrees C and 15 degrees C. The first four residues of the peptide are in a largely extended conformation. Calculations suggest that residues 5 through 14 are significantly helical at 5 degrees C. When the temperature is increased, circular dichroism spectra indicate that the helical content decreases. At 15 degrees C, the 3JN alpha coupling constants increase in the helical region, indicating an increase in motion or conformational averaging in the helical segment. None of the peptides has pH titration behavior consistent with salt bridge stabilization of helical conformation. Our data lend themselves to interpretation with the helix dipole model and specific side-chain interactions. When the N and C termini charges are removed the helical content of the peptides increases. The amount of helicity increases as the pH is lowered, due to the ionization of His16. Much of the helical stabilization appears to be due to a specific side-chain interaction between His16 and Tyr12.     

Molecular conformation of alpha-bungarotoxin as studied by nuclear magnetic resonance and circular dichroism

We have examined the circular dichroism and nuclear magnetic resonance spectra of a long neurotoxin, alpha-bungarotoxin, over a wide range of pH values and temperatures, and under high salt conditions. The observations are interpreted partly in terms of the known crystal structure of this polypeptide. We support earlier findings of a greater degree of beta-sheet structure in solution than has been reported by X-ray crystallography and, importantly, the invariant residue associated with neurotoxicity, Trp29, is shown to be in a similar environment to that found in alpha-cobratoxin and LS III from Laticauda semifasciata. The implications of this observation for structure/function relationships are outlined.     

13C nuclear magnetic resonance and circular dichroism studies of the tuftsin conformation in water

13C-NMR and circular dichroic (CD) spectra of tuftsin and its analogues are discussed in connection with our hypothesis that the beta-turn is the biologically active conformation of tuftsin. The changes in CD spectra evoked by an increase in pH are interpreted as a demonstration of the increasing amount of beta-turn conformers in solution. Configurational changes in successive residues of tuftsin showed that residues 2 and 3 of the peptide chain are important for the tuftsin conformation.     

Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).     

Solution structure of human growth hormone releasing factor. Combined use of circular dichroism and nuclear magnetic resonance spectroscopy

The solution structures of two human growth hormone releasing factor analogues, 27Leu45Gly-hGHRF(1-45)OH and 27Nle-hGHRF(1-29)NH2, are investigated by means of circular dichroism and nuclear magnetic resonance spectroscopy. Using circular dichroism spectroscopy, it is shown that both peptides adopt ordered structures at low concentrations of trifluoroethanol (approximately 30%). Quantitative analysis of the circular dichroism spectra indicates that the same number of residues, approximately 23 to 25, are in a helical state in both peptides. Using two-dimensional nuclear magnetic resonance methods all proton resonances of the 27Nle-hGHRF(1-29)NH2 fragment are assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. Two distinctive regions of alpha-helix are present extending from residues 6 to 13 and 16 to 29.     

A 13C nuclear magnetic resonance and circular dichroism study of the collagen-gelatin transformation in enzyme solubilized collagen.

Natural abundance Fourier transform 13C nuclear magnetic resonance (13C NMR) were obtained for enzyme solubilized collagen at 1 degrees intervals through the transition region. The transition of collagen molecules from the rigid triple helical state to single-stranded, random-coil state is accompanied by a change from broadened carbon resonances unobservable under high-resolution conditions to narrow line spectra. Thus distinction can be made between helical and random-coil states of individual residues. The transition is monophasic, as determined by examination of 14 different carbon resonances, and the entire structure is found to melt cooperatively over a temperature interval of 5 +/- 1 degrees. All the residues seem to be involved in the unfolding process concurrently. The transition was also studied by examining the changes in the circular dichroism spectrum brought about by heating. The experiments corroborated the observation that the transition proceeded cooperatively over a temperature interval of 4 degrees. Enzyme soluble collagen is seen to melt less cooperatively than native collagen. The enthalpy change was determined by assuming an equilibrium between three random coil gelatin chains and tropocollogen molecules. From the enthalpy, the average length of the tripeptide sequences (70-85) involved in the transition can be estimated. The shortening of the cooperative unit could arise as a result of some alteration of the native conformation through proctase treatment.     

Frequency domain magnetic resonance and magnetic circular dichroism studies on Ni4 cubane molecular nanomagnets: A magnetic anisotropy investigation

A study of the magnetic anisotropies of three Ni cubane single-molecule magnets (SMM), [Ni(hmp)(MeOH)Cl]₄·H₂O (1·H₂O) (hmpH = (2-hydroxymethyl)pyridine) [Ni(hmp)(dmb)Cl]₄ (2) (dmb = 3,3-dimethylbutanol) and [Ni(hmp)(dmp)Cl]₄ (3) (dmp = 2,2-dimethylpropanol) is reported. Frequency domain magnetic resonance spectroscopic (FDMRS) studies on 1, 2 and 3 as powder pellets reveal zero-field splitting (ZFS) for the spin ground states of these compounds in the solid state. The ZFS of the complexes 1 and 2 were determined and the presence of different molecular species was found in both complexes while only one species was found in 3. The nesting of the variable temperature variable field (VTVH) curves observed from magnetic circular dichroism (MCD) measurements on 1 in solution confirms the presence of ZFS. Virtually all the bands observed in the magnetic circular dichroism spectra possess the same sign, which may be related to the ferromagnetic exchange coupling. In addition, MCD measurements of a dilute solution demonstrate the molecular origin of the magnetic anisotropy in 1.     

Conformations of fibroblast and E. coli-derived recombinant human interferon-beta s as studied by nuclear magnetic resonance and circular dichroism

The conformations of fibroblast and E. coli-derived recombinant human interferon-beta s were studied by circular dichroism and nuclear magnetic resonance spectroscopy in the acidic pH region of 4.6 to 1.6. Both interferons have very similar conformations with high alpha-helix contents (approximately 70%). These results suggest that glycosylation does not appreciably change the conformation of human interferon-beta. Moreover, a slow conformational change is observed below pH 2.0, which induces the disruption of beta-sheets.     

Conformation of the RNA polymerase II C-terminal domain: circular dichroism of long and short fragments

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandemly repeated copies of a heptapeptide with the Y(1)S(2)P(3)T(4)S(5)P(6)S(7) consensus sequence. This repeat contains two overlapping SPXX motifs that can adopt a beta-turn conformation. In addition, each CTD repeat contains the PXXP sequence characteristic of the left-handed helix of polyproline II (P(II)) found in SH3 domain ligands and the PXY sequence that is the target for WW domains. We have studied CTD fragments using circular dichroism (CD) to characterize the conformation of the CTD in water and in the hydrogen bond-promoting solvent trifluoroethanol (TFE). In water, an eight-repeat fragment is predominantly unordered, but at 32 degrees C has P(II) and beta-turn contents estimated to be about 15 % and less than 10 %, respectively. In 90 % TFE, the beta-turn fraction is estimated to be about 75 %, the remainder being unordered and P(II) conformations. The Tyr side-chains are ordered to a significant extent in 90 % TFE. Replacement of the fully conserved Pro residues by alpha-aminoisobutyric acid leads to a large increase in beta-turn. Replacement of Ser2 by Ala does not substantially alter the CTD conformation in water or TFE. Ser5 replacement by Ala increases the P(II) content in water and affects the conformation in TFE-rich solutions. Phosphorylation of Ser2 and Ser5 has little effect in water, but Ser2 affects the conformation in TFE-rich solution in much the same way as Ser5-->Ala substitution. The CD of the full-length murine CTD in water is similar to that of the eight-repeat fragment, indicating little difference in conformation with increasing chain length beyond eight repeats. The roles of P(II) and beta-turn in the interaction of CTD with its target proteins (mediator and RNA-processing components) are discussed. The most likely interactions are between P(II) and WW or SH3 domains, or with some unknown P(II)-binding motif.     

Circular dichroism and magnetic circular dichroism of bismuth-induced,metallothionein-like proteins

Optical studies have been carried out on bismuth-containing proteins which were isolated from the livers and kidneys of rats following injections of BiCl₃. Absorption, circular dichroism and magnetic circular dichroism spectra of hepatic Bi,Zn-metallothionein 1 and 2 indicate that the spectra are dominated by transitions from the zinc thiolate chromophore. The data from the renal Bi,Cu-metallothionein 2 are quite different and it is suggested that these spectra involve a mixture of transitions from the bismuth and copper thiolate binding sites.     

The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies.

The segment 32-47 of the N-terminal extracellular domain of the type A cholecystokinn receptor, CCK(A)-R(32-47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCK(A)-R(32-47) encompassing residues 39-46 adopted a well-defined secondary structure in the presence of DPC micelles, whereas the conformation of the N-terminal region (segment 32-37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCK(A)-R(32-47) was different from that found for the Intact N-terminal receptor tail, CCK(A)-R(1-47). To assess whether CCK(A)-R(32-47) was still able to bind the nonsulfated cholecystokinin C-terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCK(A)-R(1-47) and CCK8. The different affinities for the ligand shown by CCK(A)-R(32-47) and CCK(A)-R(1-47) were paralleled by different interaction modes between the receptor segments and the micelles.The interaction of CCK(A)-R(32-47) with DPC micelles was much weaker than that of CCK(A)-R(1-47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCK(A)-R(32-47] was found to form non-micellar adducts with the detergent that prevented the onset of a functionally significant Interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1-31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N-terminal extracellular tail of the CCKA receptor.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Role of the conserved acidic residue Asp21 in the structure of phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and nuclear magnetic resonance studies

To elucidate a role of the Src homology 3 (SH3)-conserved acidic residue Asp21 of the phosphatidylinositol 3-kinase (PI3K) SH3 domain, structural changes induced by the D21N mutation (Asp21 --> Asn) were examined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. In the previous study, we demonstrated that environmental alterations occurred at the side chains of Trp55 and some Tyr residues from the comparison of the near-UV CD spectra of the PI3K SH3 domain with or without a D21N mutation [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr14 and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue with or without a D21N mutation. The (1)H and (15)N resonance assignments of the PI3K SH3 domain and its D21N mutant revealed that significant chemical shift changes occurred to the aromatic side-chain protons of Trp55 and Tyr14 upon the D21N mutation. All these aromatic residues are implicated in ligand recognition. In addition, the NMR analysis showed that the backbone conformations of Lys15-Asp23, Gly54-Trp55, Asn57-Gly58, and Gly67-Pro70 were affected by the D21N mutation. Furthermore, the (15)N[(1)H] nuclear Overhauser effect values of Tyr14, Glu19, and Glu20 were remarkably changed by the mutation. These results show that the D21N mutation causes structural deformation of more than half of the ligand binding cleft of the domain and provide evidence that Asp21 plays an important role in forming a well-ordered ligand binding cleft in cooperation with the RT loop (Lys15-Glu20).     

Conformation of malformin A: a proton magnetic resonance and a circular dichroism study

Malformin A is a cyclic pentapeptide with an intramolecular disulfide bridge. The conformation in solution of this molecule has been studied by NMR and CD. The 270 MHz Proton spectrum in dimethyl sulfoxide is well resolved and the peaks corresponding to the five residues have been assigned. From the temperature dependence of chemical shifts of the peptide protons and from the exchange rate of these protons, it is concluded that the NH proton of one Cys is shielded from the solvent. This observation and H? N? _αC? H angles, estimated from the corresponding coupling constants, a proposed conformation of the peptide backbone. From the H? _βC? _αC? H coupling constants, a P chirality for the disulfide bridge is proposed. Such a conformation is confirmed by the circular dichroism spectrum which shows a negative band at λ > 250 nm. It is concluded that the conformation of malformin A is rigid and that the disulfide bridge is exposed to interact with biological receptors.     

I. Study of protein aggregation due to heat denaturation: A structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering

The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, Tm, of approximately 65 degrees C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around Tm using near- and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements. These measurements revealed the presence of folded structures at 55 degrees C and below, while denatured structures appeared at 65 degrees C and above. Concurrent static light scattering (SLS) measurements, employed to detect the presence of RNase A aggregates, showed that RNase A aggregation was observed at 65 degrees C and above, when much of the protein was denatured. Subsequent NMR time-course data demonstrated that aggregates forming at 75 degrees C and pH 7.8 were indeed derived from heat-denatured protein. However, aggregation was also detected at 55 degrees C when the spectroscopic data suggested the protein was present predominantly in the folded configuration. In contrast, heat denaturation did not lead to RNase A aggregation in a very acidic environment. We attribute this phenomenon to the effect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH.     

Conformational requirement of signal sequences functioning in yeast: circular dichroism and 1H nuclear magnetic resonance studies of synthetic peptides

Recently, we have designed a series of simplified artificial signal sequences and have shown that a proline residue in the signal sequence plays an important role in the secretion of human lysozyme in yeast, presumably by altering the conformation of the signal sequence [Yamamoto, Y., Taniyama, Y., & Kikuchi, M. (1989) Biochemistry 28, 2728-2732]. To elucidate the conformational requirement of the signal sequence in more detail, functional and nonfunctional signal sequences connected to the N-terminal five residues of mature human lysozyme were chemically synthesized and their conformations in a lipophilic environment [aqueous trifluoroethanol (TFE) or sodium dodecyl sulfate micelles] analyzed by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. The helix content of the peptides, including functional (L8, CL10) and nonfunctional (L8PL, L8PG, L8PL2) signal sequences, was estimated from CD spectra to be 40-50% and 60-70%, respectively, indicating that the helical structure is more abundant in the nonfunctional signal sequences. Two-dimensional NMR analyses in 50% TFE/H2O revealed that each peptide adopted a helical conformation throughout the sequence except for a few residues at the N- and C-termini. Furthermore, H-D exchange experiments indicated that the helical structure of the C-terminal region of the functional signal sequences (L8 and CL10) was less stable than that of the nonfunctional signal sequences (L8PL and L8PL2). On the basis of these results, a model was developed in which the functional signal sequence is inserted in the membrane with a helical conformation and the C-terminal helix unraveled in an extended conformational form through an interaction with the signal peptidase.