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).     

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.     

Structure,stability, and function of hDim1 investigated by NMR,circular dichroism,and mutational analysis

The 142 amino acid Dim1p protein is a component of the U4/U6.U5 tri-snRNP complex required for pre-mRNA splicing and interacts with multiple splicing-associated proteins. To gain further insight into the structural basis of its function, we determined the solution structure of the reduced form of the dominant negative human hDim1 (hDim1(1)(-)(128)) using multidimensional NMR spectroscopy. This dominant negative hDim1 assumes a thioredoxin-like fold, confirming previous NMR and crystallographic results. However, in contrast to a recent crystal structure, the NMR solution structure for the reduced form of hDim1(1)(-)(128) presented here, along with thermodynamic data, indicates that the presence of a disulfide bond between Cys38 and Cys79 is structurally and functionally unimportant. Comparison of the truncated hDim1(1)(-)(128) with the full-length protein, using NMR and circular dichroism spectroscopy, indicates that the 14 C-terminal residues can undergo a local unfolding transition and assume alternative conformations, which appear to play a functional role. Other residues essential for hDim1 function are identified by using mutational and genetic approaches. The residues thus identified are not identical with those previously shown to govern Dim1 interaction with defined protein partners.     

Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy.

The kinetics of protein folding for horse ferricytochrome c was investigated by stopped-flow methods, using far-UV circular dichroism (CD), near-UV CD, and tryptophan fluorescence to probe the formation of secondary structure and tertiary interactions. In the far-UV region of the CD spectrum (222 nm), 44% of the total change associated with refolding occurs within the dead time of the stopped-flow experiment, indicating that a significant amount of helical secondary structure is formed in less than 4 ms. The remaining changes in the ellipticity at 222 nm occur in two kinetic phases with time constants of about 40 ms and 0.7 s, respectively. In contrast, there is no evidence for rapid changes in the ellipticity at 289 nm: an aromatic CD band, which is indicative of the formation of a tightly packed core, only begins to appear in a 400-ms step and is completed in a final 10-s phase. The fluorescence of a single tryptophan at position 59, which becomes quenched upon folding via nonradiative energy transfer to the heme group, provides complementary information on the condensation of the polypeptide chain during refolding. The fluorescence-detected stopped-flow folding kinetics of ferricytochrome c exhibits a 35% decrease in fluorescence during the dead time, suggesting that a substantial decrease in the average tryptophan-heme distance occurs on a submillisecond time scale. The subsequent fluorescence changes exhibit two prominent phases with time constants of about 20 and 300 ms, followed by a minor 5-s phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

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.     

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.     

Characterization of the cadmium(II) binding site in Cd,Zn-metallothionein by magnetic circular dichroism spectroscopy

Absorption and magnetic circular dichroism spectra of rat liver Cd, Zn-metallothionein, and the cadmium complexes of propanethiolate and 1,2 propanedithiolate are reported. Observation of the same derivative-like MCD signal in the 250 nm region of each of these species provides experimental evidence for the assignment of the 250 nm shoulder in the Cd, Zn-metallothionein absorption spectrum as a sulfur to cadmium charge transfer band.     

Kreb's TCA cycle in Halobacterium salinarum investigated by 13C nuclear magnetic resonance spectroscopy

Kreb's tricarboxylic (TCA) cycle was studied in Halobacterium salinarum cells grown in the presence of glucose or alanine. The cells were incubated with ¹³C-labeled substrate and the labeling pattern of various carbon positions in glutamate was monitored by ¹³C-NMR spectroscopy. [2-¹³C]pyruvate, when used as a substrate, led mainly to signals for C-1 and C-5 glutamate, with some C-3 glutamate. [3-¹³C]pyruvate as a substrate produced signals, mainly C-2, C-3, and C-4 glutamate, with some C-1 and C-5 glutamate. The multiplicity of the signals and observation of a C-1 signal in this case indicates extensive cycling of the label in the TCA cycle. Isotopomer analysis of glutamate labeling suggested that of the total pyruvate entering the TCA cycle, the flux through pyruvate:ferredoxin oxidoreductase was 90% while that through pyruvate caboxylase was 10%. Only 53% of the total acetyl-CoA was produced from the added labeled pyruvate, the rest being generated endogenously. In the presence of nitrogen, mainly transamination reaction products were formed in the case of both these substrates. Received: November 26, 1997 / Accepted: May 11, 1998     

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.     

Conformations of alanine-based peptides in water probed by FTIR, Raman, vibrational circular dichroism, electronic circular dichroism, and NMR spectroscopy

We have used a combination of FTIR, VCD, ECD, Raman, and NMR spectroscopies to probe the solution conformations sampled by H-(AAKA)-OH by utilizing an excitonic coupling model and constraints imposed by the 3JCalphaHNH coupling constants of the central residues to simulate the amide I' profile of the IR, isotropic Raman, anisotropic Raman, and VCD spectra in terms of a mixture of three conformations, i.e., polyproline II, beta-strand and right-handed helical. The representative coordinates of the three conformations were obtained from published coil libraries. Alanine was found to exhibit PPII fractions of 0.60 or greater, mixed with smaller fractions of helices and beta-strand conformations. Lysine showed no clear conformational propensity in that it samples polyproline II, beta-strand, and helical conformations with comparable probability. This is at variance with results obtained earlier for ionized polylysine, which suggest a high polyproline II propensity. We reanalyzed previously investigated tetra- and trialanine by combining published vibrational spectroscopy data with 3JCalphaHNH coupling constants and obtained again blends dominated by PPII with smaller admixtures of beta-strand and right-handed helical conformations. The polyproline II propensity of alanine was found to be higher in tetraalanine than in trialanine. For all peptides investigated, our results rule out a substantial population of turn-like conformations. Our results are in excellent agreement with MD simulations on short alanine peptides by Gnanakaran and Garcia [(2003) J. Phys. Chem. B 107, 12555-12557] but at variance with multiple MD simulations particularly for the alanine dipeptide.     

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.     

Influence of Ca2+ binding on the structure and stability of bovine alpha-lactalbumin studied by circular dichroism and nuclear magnetic resonance spectra

Both the Ca2+-bound and Ca2+-free forms of alpha-lactalbumin can assume essentially the same folded conformation as evidenced by similarity in their CD and proton n.m.r. spectra. Thermal unfolding followed by the aromatic CD has shown that the stability of the folded state is markedly enhanced by Ca2+ and that the stabilization is almost entirely entropic; addition of 0.1 mM Ca2+ shifts the transition temperature from 40 degrees to 62 degrees in 0.1M Na+ at pH 7.0. The enthalpy change of the unfolding, coincident between the two forms, is, however, significantly smaller than that known for lysozyme. The n.m.r. spectrum under the condition that both the forms of the protein are in the folded state reflects minor environmental changes of certain protons upon Ca2+ binding, and these changes are shown to afford useful probes for assessment of the location of the binding site. From the pH dependence and temperature dependence of the spectrum and also by using spin decoupling in the aromatic region (6.4-8.7 p.p.m.), it is shown that none of histidyl residues are affected and that at least two tryptophanyl ring protons experience environmental changes upon Ca2+ binding to the folded apo-protein. Effect of free excess Ca2+ on the spectrum has also shown that in native alpha-lactalbumin there is only one Ca2+-binding site that is detectable by the present method.     

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.     

Noninvasive quantitative in vivo mapping and metabolism of boronophenylalanine (BPA) by nuclear magnetic resonance (NMR) spectroscopy and imaging

10B-enriched L-p-boronophenylalanine (BPA) is one of the compounds used in boron neutron capture therapy (BNCT). In this study, several variations of nuclear magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) were applied to investigate the uptake, clearance and metabolism of the BPA-fructose complex (BPA-F) in normal mouse kidneys, rat oligodendroglioma xenografts, and rat blood. Localized 1H MRS was capable of following the uptake and clearance of BPA-F in mouse kidneys with temporal resolution of a few minutes, while 1H MRSI was used to image the BPA distribution in the kidney with a spatial resolution of 9 mm3. The results also revealed significant dissociation of the BPA-F complex to free BPA. This finding was corroborated by 1H and 11B NMR spectroscopy of rat blood samples as well as of tumor samples excised from mice after i.v. injection of BPA-F. This investigation demonstrates the feasibility of using 1H MRS and MRSI to follow the distribution of BPA in vivo, using NMR techniques specifically designed to optimize BPA detection. The implementation of such procedures could significantly improve the clinical efficacy of BNCT.     

Characterization of selectively 13C-labeled synthetic melittin and melittin analogues in isotropic solvents by circular dichroism, fluorescence, and NMR spectroscopy

The spectroscopic and functional characterization of 13C-labeled synthetic melittin and three analogues is described. Selectively 13C-enriched tryptophan ( [13C delta 1]-L-Trp) and glycine ( [13C alpha]Gly) were incorporated into melittin and three analogues by de novo peptide synthesis. 13C-Labeled tryptophan was incorporated into melittin at position 19 and into single-tryptophan analogues of melittin at positions 17, 11, and 9, respectively. Each of the synthetic peptides contained 13C-labeled glycine at position 12 only. The peptides were characterized functionally in a cytolytic assay, and spectroscopically by CD, fluorescence, and NMR. The behavior of 13C-labeled synthetic melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. All of the analogues were found to be efficient lytic agents and thus were functionally similar to the native peptide, yet no evidence was found for formation of a melittin-like tetramer by any of the analogues in aqueous media, although there was a propensity for apparently nonspecific peptide aggregation, especially for MLT-W9. Since the analogues did exhibit fractional helicities by CD comparable to or even greater than melittin itself in the presence of methanol, we infer that tetramer assembly requires not only the ability to form alpha-helix but also a very precise packing of amino acid side chains of the constituent monomers. The 13C chemical shift of the Gly-12 C alpha was found to be a sensitive marker for helix formation in all of the peptides. For melittin itself, 13C NMR spectra revealed a downfield shift of approximately 1.8 ppm for the Gly-12 13C alpha resonance of the tetramer relative to that observed for the free monomer in D2O. In mixed samples containing melittin monomer and tetramer, two discrete Gly-12 13C alpha peaks were observed simultaneously, suggestive of slow exchange between the two species. We conclude that melittin's ability to form a soluble tetramer is not a prerequisite for cytolytic activity, nor is cytolytic potential precisely correlated with the ability to form an amphiphilic helix.