Double-headed protease inhibitors from black-eyed peas. I. Purification of two new protease inhibitors and the endogenous protease by affinity chromatography.

Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.     

Double-headed protease inhibitors from black-eyed peas. V. Analysis of the energetics of protease-inhibitor interactions.

The half-site reactivity of trypsin and chymotrypsin binding to two double-headed black-eyed pea protease inhibitors a trypsin-chymotrypsin inhibitor (BEPCI) had a trypsin inhibitor (BEPTI), is explained in terms of the energetics of these inhibitor-protease interactions. Free energy diagrams are constructed to facilitate interpretation of the energetics. Coupling-free energies are calculated to reflect the magnitude of the interdependence of protease-binding (alloassociation) and inhibitor subunit interactions (isoassociation). The experiment observation of the predominance of liganded monomer complexes for the lima bean inhibitor and the Bowman-Birk soybean inhibitor and the predominance of half-site-liganded complexes for BEPCI and BEPTI is the direct result of the magnitudes and signs of the coupling free energies which result from these protease-inhibitor interactions.     

Double-headed protease inhibitors from black-eyed peas. VI. Singlet-singlet energy transfer and other optical studies on the structure of trypsin and chymotrypsin complexes.

The geometry of the binary and ternary complexes of two black-eyed pea inhibitors with trypsin and chymotrypsin has been established by distance measurements using the technique of singlet-singlet energy transfer. Triangulation of measured distances in the ternary double-headed complex of the trypsin-chymotrypsin inhibitor (BEPCI) with trypsin and chymotrypsin limits the possible structural models for this complex to those in which the center to center distance between trypsin and chymotrypsin is about 64 A, the distance from the center of trypsin to the single fluorescently labeled tyrosyl residue of the BEPCI dimer is about 33 A, and the distance between the chymotrypsin center and the labeled tyrosine of the inhibitor is about 43 A. Energy transfer results for the trypsin inhibitor (BEPTI) complexes show conclusively that the weak trypsin site is structurally analogous to the strong chymotrypsin binding site of BEPCI. The weak chymotrypsin binding site of BEPTI is structurally analogous to the strong trypsin sites of BEPCI and BEPTI. Corresponding distances in binary and ternary complexes are the same, indicating that little or no structural rearrangement occurs when the ternary complexes are formed. Complex formation was shown to involve tryptophan and tryosine residues of both trypsin and chymotrypsin as judged by absorption and circular dichroism difference spectroscopy. In addition, circular dichroism difference spectra revealed some disulfide contributions.     

Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin.

Complex formation between two new double-headed protease inhibitors from black-eyed peas, trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI), and trypsin and chymotrypsin was investigated in the concentration range from 10-8 to 10-4 M by titration experiments and gel filtration chromatography. Dissociation equilibrium constants measured for complexes detected in the titration experiments range from as large as 10-8 M for trypsin bound nonspecifically to the chymotrypsin site of BEPCI to as small as 10-18 M2 for the interaction of BEPCI with chymotrypsin. The identity and stoichiometry of complexes detected during titration experiments were confirmed by gel filtration of mixtures of native and fluorescently labeled proteases and inhibitors. Half-site reactivity is observed in the formation of complexes between BEPCI or BEPTI and trypsin and chymotrypsin at all experimentally practical concentrations. The double-headed complex contains 1 molecule each of trypsin, chymotrypsin, and BEPCI dimer. The bimolecular rate constants of complex formation between trypsin or chymotrypsin and isolated BEPCI oligomers range from 1.8 X 10(5) M-1 S-1 for chymotrypsin and BEPCI monomer to 4.4 X 10(7) M-1 S-1 for trypsin and the rapidly equilibrating BEPCI dimer. The estimated rate constants for the dissociation of half-site-liganded dimer complexes and liganded monomer complexes range from 7.5 X 10-3 S-1 for the trypsin-liganded BEPCI monomer complex to 1.6 X 10-6 S-1 for the chymotrypsin-liganded BEPCI dimer complex.     

Double-headed protease inhibitors from black-eyed peas. III. Subunit interactions of the native and half-site chemically modified proteins.

The chemical modification of two new double-headed-protease inhibitors from black-eyed peas, a trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI) with dansyl chloride was investigated under various conditions. The NH2-terminal serine of both BEPCI and BEPTI, the 4 lysyl residues of BEPCI, and 4 of the 5 lysyl residues of BEPTI, could not be dansylated in the absence of urea. The single tyrosine per subunit of BEPCI and BEPTI was unreactive even in the presence of urea but could be labeled with half-site reactivity by the Celite method. Lysine, NH2-terminal serine, and tyrosine were reactive in fully reduced, carbamidomethylated BEPCI and BEPTI. Gel filtration was used to study the subunit interactions of BEPCI and BEPTI. At pH 8 or pH 3.0 there is a complex set of multiple equilibria with widely differing rates of attainment. We have found evidence for a rapid dimer-tetramer equilibrium, a distinct moderate rate dimer-tetramer equilibrium, a very slow monomer-dimer equilibrium, and postulate slow isomerization of the two forms of dimer and the two forms of tetramer. The monomer-dimer equilibrium is quite unusual in that the dimer is stabilized by chaotropic ions and even slightly by guanidine HC1. In contrast to the complex pattern seen in native BEPCI, the half-site, dansylated BEPCI exists at similar concentration exclusively as a tetramer at neutral pH.     

Structural studies on prolyl hydroxylase. Conformation from circular dichroism spectroscopy

The circular dichroism spectra of purified prolyl hydroxylase [EC 1.14.11.12] in the native and heat-denatured states were obtained at pH 7.8. Analysis of the far-uv spectrum of the native enzyme gave an estimate of 40% alpha-helix, 40% beta-structure and 20% random coil. The near-uv spectrum contained several negative dichroic bands that can be attributed to phenylalanyl, tyrosyl and tryptophyl residues situated in an asymmetric environment in the protein. These bands were not seen in the enzyme denatured by heat. The denaturation was monitored by changes in the alpha-helical content as well as the activity of the enzyme as a function of temperature. The normalized transition profiles with respect to the change in helical content and the loss of enzyme activity were coincidental, indicating the involvement of the alpha-helical segments in maintaining the enzyme activity.     

Structural studies of vinblastine alkaloids by exciton coupled circular dichroism

SCF-CI-dipole velocity MO calculations have shown that the bisignate circular dichroic curves of vinblastine/vincristine alkaloids at ca 210 and 220–230 nm are due to exciton coupling between the indoline and indole moieties. Furthermore, a combination of X-ray crystal structure data with MM2 local energy minimization provides a convenient means for estimation of the preferred solution conformation.     

Conformational studies on parvalbumins by circular dichroism.

Structural variations of two parvalbumins, Whiting III and Pike III, in various denaturing conditions, have been studied by circular dichroism. CD signals are depressed from 4 urea. For Pike III, acidic pH, sodium dodecyl sulfate or complete removal of Ca2+ show little effect in the far ultraviolet region but rather strong effects in the near ultraviolet. For Whiting III similar results are obtained at acidic pH. Carboxymethylated Whiting III (0.15 Ca2+/mol) shows, on the contrary, decreased CD signals in the far and in the near ultraviolet spectra. Addition of Ca2+ fully restores the native CD spectra in both proteins. Ca2+ binding produces structural modifications which are found to vary according to parvalbumin and which seem in any case different from those described for troponin C.     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. II. A DeVoe theory approach.

The DeVoe polarizability theory is used to calculate vibrational circular dichroism (VCD) and infrared (IR) absorption spectra of four polyribonucleotides: poly(rA) x poly(rU), poly(rU) x poly(rA) x poly(rU), poly(rG) x poly(rC), and poly(rC+) x poly(rI) x poly(rC). This is the first report on the use of the DeVoe theory to calculate VCD, oriented VCD, IR absorption, and IR linear dichroism (LD) spectra of double- and triple-stranded polyribonucleotides. Results are reported for DeVoe theory calculations--within the base-stretching 1750-1550 cm(-1) spectral region--on several proposed multistranded polyribonucleotide geometries. The calculated spectra obtained from these proposed geometries are compared with previously reported measured and calculated VCD and IR spectral results. Base-base hydrogen-bonding effects on the frequencies and magnitudes of the base carbonyl stretching modes are explicitly considered. The good agreements found between calculated and measured spectra are proposed to be further evidence of the usefulness of the DeVoe theory in drawing three-dimensional structural conclusions from measured polyribonucleotide VCD and IR spectra.     

Spectra of circular dichroism of proteinase inhibitors from corn

The circular dichroism spectra of trypsin and chymotrypsin inhibitors from corn seeds were investigated. The spatial organization of the inhibitor molecules was shown to be different. The effects of guanidine chloride and temperature on these proteins suggest that the trypsin inhibitor from corn is much more stable towards the denaturating influences than the chymotrypsin inhibitor.     

Tryptophan mutants of intestinal fatty acid-binding protein: ultraviolet absorption and circular dichroism studies.

An UV absorption and CD study of intestinal fatty acid-binding protein is presented. Since there are only two Trp residues in the molecule, two single-Trp mutants were prepared to deconvolute their signals. The individual contribution of the eight Phe and four Tyr residues was not established; however, Phe global contribution is relatively free of interferences from the other chromophores and was observed directly. CD spectra showed that Phe vibronic structure was unusually sharp and seems to monitor very specific details in the three-dimensional structure. The global signal from Tyr was assigned only approximately due to band broadening and overlapping. At the upper end of the CD spectrum, strong positive (1)L(b) Trp transitions from Trp 82 and strong negative (1)L(b) Trp transitions from Trp 6 were observed. (1)L(a) transitions were overall weak, positive for Trp 82 and negative for Trp 6, nearly cancelling each other out in the final spectrum. The above assignment is of practical and fundamental interest to monitor folding, binding, and molecular dynamics down to microdomain resolution. The assignment of Trp bands allowed comparison with previous data from CRABP1, another member of the IFABP family with 28% identical residues. It was found that structural homology extends beyond sequence and tertiary fold to include optical properties of equivalent Trp residues in the structure.     

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.     

Protein conformational relaxation following photodissociation of CO from carbonmonoxymyoglobin: picosecond circular dichroism and absorption studies

Picosecond time-resolved polarization spectroscopy is used to study relaxation dynamics in myoglobin following photoelimination of CO from carbonmonoxymyoglobin. Evolution of the transient circular dichroism signal of the N band of myoglobin (probed at 355 nm) to that characteristic of equilibrium myoglobin requires approximately 300 ps. This time scale is significantly longer than that corresponding to the photoinitiated bond cleavage. Transient linear dichroism of the Soret band and picosecond time-resolved magnetic circular dichroism measurements of the Q band demonstrate that the circular dichroism kinetics do not result from either time-dependent changes in the orientation of the transition moments of the heme ring or the doming of the heme that accompanies the out-of-plane motion of the iron. Finally, transient absorption data of the near-IR optical transition of photogenerated myoglobin suggest that the circular dichroism data are not a measure of the tilting of the proximal histidine. The circular dichroism data are discussed in terms of a relaxation in the tertiary structure of the protein following dissociation.     

Structural similarity of chymopapain forms as indicated by circular dichroism.

Four chymopapain forms were isolated by high-resolution liquid chromatography on a cation-exchange column. The three major forms possess nearly identical secondary and tertiary structures, as judged from their c.d. spectra; these components showed similar proteolytic activity and Mr values close to that of papain. The fourth isolated component seems to be a mixture of modified proteins.     

Time-resolved circular dichroism and absorption studies of the photolysis reaction of (carbonmonoxy)myoglobin.

Time-resolved circular dichroism (TRCD) and absorption spectroscopy are used to follow the photolysis reaction of (carbonmonoxy)myoglobin (MbCO). Following the spectral changes associated with the initial loss of CO, a subtle change is observed in the visible absorption spectrum of the Mb product on a time scale of a few hundred nanoseconds. No changes are seen in the CD spectrum of Mb in the visible and near-UV regions subsequent to the loss of CO. The data suggest the existence of an intermediate found after ligand loss from MbCO that is similar in structure to the final Mb product.     

Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.

The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.