Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E

Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms. When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000). Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form. The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s). The two chains are presumed to have different functions. NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188). We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography. The technique, with minor modifications, is a generalized method for types A, B, and E. These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions. The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule. This affirms that NT is composed of two chains. The two subunit chains are now usable for amino acid sequence and other studies. Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains. Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain.     

Proteolytic Cleavage of Tetanus Toxin Increases Activity

Tetanus toxin is initially synthesized in the form of a single polypeptide chain and then proteolytically "nicked" by the bacteria to produce a two-chain structure joined by a disulfide bond. This two-chain form of the toxin is the form known to be biologically active. Whether such nicking is necessary for activity, as it is for certain other bacterial toxins, has not been demonstrated previously. Single-chain toxin preparations produced by salt extraction from the bacteria are characterized and compared with pure two-chain toxin obtained from extracellular filtrates. The ability of these various toxin preparations to produce paroxysmal activity in mouse spinal cord neurons grown in dissociated cell culture is described. The pure two-chain toxin is demonstrated to have greater activity than the single-chain toxin preparations. Indeed the activity of the single-chain toxin preparations can be explained by the small amounts of residual two-chain toxin present in these extracts. Using a protease from a toxin-minus strain of Clostridium tetani to convert a single-chain toxin preparation to two-chain toxin increases toxin activity. In vivo the single-chain toxin preparation is also less toxic. These findings indicate that proteolytic nicking of tetanus toxin increases activity. The unnicked, single-chain form of tetanus toxin may be a relatively nontoxic protoxin form of the toxin; this is a structure-function relationship similar to that of other bacterial protein toxins.     

Enzymatic and fluorescence studies of four single-tryptophan mutants of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase.

In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.     

Cofactor and tryptophan accessibility and unfolding of brain glutamate decarboxylase

Cofactor and tryptophan accessibility of the 65-kDa form of rat brain glutamate decarboxylase (GAD) was investigated by fluorescence quenching measurements using acrylamide, I-, and Cs+ as the quenchers. Trp residues were partially exposed to solvent. I- was less able and Cs+ was more able to quench the fluorescence of Trp residues in the holoenzyme of GAD (holoGAD) than the apoenzyme (apoGAD). The fraction of exposed Trp residues were in the range of 30-49%. In contrast, pyridoxal-P bound to the active site of GAD was exposed to solvent. I- was more able and Cs+ was less able to quench the fluorescence of pyridoxal-P in holoGAD. The cofactor was present in a positively charged microenvironment, making it accessible for interactions with anions. A difference in the exposure of Trp residues and pyridoxal-P to these charged quenchers suggested that the exposed Trp residues were essentially located outside of the active site. Changes in the accessibility of Trp residues upon pyridoxal-P binding strongly supported a significant conformational change in GAD. Fluorescence intensity measurements were also carried out to investigate the unfolding of GAD using guanidine hydrochloride (GdnHCl) as the denaturant. At 0.8-1.5 M GdnHCl, an intermediate step was observed during the unfolding of GAD from the native to the denatured state, and was not found during the refolding of GAD from the denatured to native state, indicating that this intermediate step was not a reversible process. However, at >1.5 M GdnHCl for holoGAD and >2.0 M GdnHCl for apoGAD, the transition leading to the denatured state was reversible. It was suggested that the intermediate step involved the dissociation of native dimer of GAD into monomers and the change in the secondary structure of the protein. Circular dichroism revealed a decrease in the alpha-helix content of GAD from 36 to 28%. The unfolding pattern suggested that GAD may consist of at least two unfolding domains. Unfolding of the lower GdnHCl-resisting domain occurred at a similar concentration of denaturant for apoGAD and holoGAD, while unfolding of the higher GdnHCl-resisting domain occurred at a higher concentration of GdnHCl for apoGAD than holoGAD.     

Molecular topography and secondary structure comparisons of botulinum neurotoxin types A,B and E

Botulinum neurotoxin (NT) serotypes A, B and E differ in microstructure and biological activities. The three NTs were examined for secondary structure parameters (-helix, -sheet, -turn and random coil content) on the basis of circular dichroism; degree of exposed Tyr residues (second derivative spectroscopy) and state of the Trp residues (fluorescence and fluorescence quantuin yield). The proteins are high in -pleated sheet content (41–44%) and low in -helical content (21–28%). About 30–36% of the amino acids are in random coils. The -sheet contents in the NTs are similar irrespective of their structural forms (i.e. single or dichain forms) or level of toxicity. About 84%, 58% and 61% of Tyr residues of types A, B, and ENT, respectively, were exposed to the solvent (pH 7.2 phosphate buffer). Although the fluorescence emission maximum of Trp residues of type B NT was most blue shifted (331 nm compared to 334 for types A and E NT, and 346 nm for free tryptophan) the fluorescence quantum yields of types A and B were similar and higher than type E. In general the NTs have similar secondary (low -helix and high -sheets) and tertiary (exposed tyrosine residues and tryptophan fluorescence quantum yield) structures. Within this generalized picture there are significant differences which might be related to the differences in their biological activities.     

Primary structure of single-chain pro-urokinase

Single-chain pro-urokinase is an inactive proenzyme form of human urokinase with a single-chain structure and a Mr of 50,000 and converted to the active two-chain form by catalytic amounts of plasmin. It was isolated from culture fluid of human kidney cells and subjected to chemical (CNBr) and proteolytic (lysyl endopeptidase) degradation. The resulting peptides were separated by reverse-phase high performance liquid chromatography and subjected to automated sequence analysis. Amino acid sequence of 85% of the 411 residues recovered in 17 peptides were found to be consistent with those of the A chain (157 amino acids) and B chain (253 amino acids) of human urokinase reported by Günzler and co-workers (Günzler, W. A., Steffens, G.J., Otting, F., Kim, S.-M., A., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 133-141; 1155-1165; Steffens, G.J., Günzler, W.A., Otting, F., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 1043-1058). It revealed the presence of Lys at position 158 in single-chain pro-urokinase through which the two polypeptide chains of human urokinase are unified into one molecule. In addition, firm evidence was found that upon activation by plasmin single-chain pro-urokinase is cleaved at the Lys-Ile bond between residues 158 and 159, resulting in the formation of a two-chain urokinase molecule held together by one disulfide linkage. These results indicate that the cleavage at the Lys-Ile bond between residues 158 and 159 is responsible for conformational change, appearance of enzyme activity and reduction of its high affinity for fibrin.     

Spectroscopic analysis of acylated and deacylated myelin proteolipid protein

The effect of covalently bound fatty acid on the conformation of the myelin proteolipid protein has been studied by ultraviolet and intrinsic fluorescence spectroscopy. With dimethyl sulfoxide used as a perturbant, the exposure of Trp and Tyr residues in various mixtures of chloroform-methanol was evaluated by difference spectroscopy of the proteolipid protein (APL) and its chemically deacylated form (d-APL). The fraction of chromophoric groups exposed increased with the proportion of chloroform with 25% of the groups exposed in 1:2 chloroform-methanol and 98% in 3:1 chloroform-methanol. These conformational changes correlate well with changes in intrinsic viscosity. Values for the deacylated form were indistinguishable from those of the acylated protein, suggesting that fatty acids do not affect protein conformation in organic solvents. In water, UV difference spectroscopy indicated that the number of Tyr and Trp groups exposed in both APL and d-APL was relatively small and was independent of the molecular size of the perturbant. However, differences in the environment of the Trp groups in the two forms of the protein could be demonstrated by intrinsic fluorescence. When the protein was excited at 295 nm, the maximum emission wavelength for the acylated protein was 330 nm, whereas it was 335 nm for the deacylated form. Furthermore, the Trp groups in d-APL were more easily quenched by acrylamide than in APL, indicating that they were more exposed, or in a more hydrophilic environment, following deacylation. Protein aggregation appears to be independent of the presence of fatty acids, suggesting that the fluorescence differences between APL and d-APL are related to factors other than aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues

We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.     

In vitro methionine oxidation of Escherichia coli-derived human stem cell factor: effects on the molecular structure, biological activity, and dimerization.

The effect of oxidation of the methionine residues of Escherichia coli-derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine-containing peptides, using RP-HPLC of Asp-N endoproteinase digests. The initial oxidation rates for Met159, Met-1, Met27, Met36, and Met48 were 0.11 min-1, 0.098 min-1, 0.033 min-1, 0.0063 min-1, and 0.00035 min-1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295-nm Trp peak in the near-UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two- and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000–10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.     

Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipoproteins of dimyristoylphosphatidylcholine

The structure of the exchangeable apolipoprotein, apolipophorin-III from Locusta migratoria, apoLp-III, is described as a bundle of five amphipathic alpha-helices. To study the interaction of each of the helices of apoLp-III with a lipid surface, we designed five single-Trp mutants, each containing a Trp residue in a different alpha-helix. The Trp residues were located in the nonpolar domains of the amphipathic alpha-helices. The kinetics of the spontaneous interaction of the mutants with dimyristoylphosphatidylcholine (DMPC) indicated that all mutants behaved as typical exchangeable apolipoproteins. Circular dichroism in the far-UV indicated that all proteins have a high and similar helical content in the lipid-bound state. The interaction of the Trp residues with the lipid surface was investigated in recombinant lipoprotein particles made with DMPC. The properties of the Trp residues were investigated by fluorescence spectroscopy. These studies showed major changes in the spectroscopic properties of the Trp residues upon binding to lipid. These changes are observed with all single-Trp mutants, indicating that a major conformational change, which affects the properties of all helices, takes place upon binding to lipid. The position of the fluorescence maximum, the quenching efficiency of acrylamide as determined by steady-state and time-resolved fluorescence, and the fluorescence lifetimes of the single-Trp mutants suggest that helices 1, 4, and 5 interact with the nonpolar domains of the lipid. The properties of the Trp in helices 2 and 3 suggest that these helices adopt a different binding configuration than helices 1, 4, and 5. Helices 2 and 3 appear to be interacting with the polar headgroups of the phospholipids or constitute a different domain that does not interact with the lipid surface.     

Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli

A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I⁻, Cs⁺ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I⁻, was proven to be an ineffective quencher with Stern-Volmer constants, K_sv, of 0.60 and 0.63 M⁻¹, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs⁺, showed about a 2-fold difference in the K_sv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25–37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs⁺ showed more than 40% increase in the K_sv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Fluorescence studies on a membrane-embedded peptide from the carboxy terminus of lipophilin

Fluorescence of an intramembranous polypeptide (T-3) derived from the carboxy-terminal sequence of lipophilin was studied in aqueous solution, detergent micelles, and lipid vesicles. In all cases, the fluorescence of the only Trp (211) was indicative of a hydrophobic, buried residue. Addition of lysophosphatidylcholine (LPC) or phosphatidylcholine (PC) gave Trp-211 a more hydrophobic, less quenching environment as compared to that in aqueous solution. Energy transfer between Trp and Tyr observed in aqueous solution was decreased by the addition of lipid or detergent. There was limited quenching by acrylamide both in the aqueous and in the lipid or detergent environments. However, PC or LPC further decreased this quenching. Cs+ and I- were even less accessible than acrylamide to Trp, further proving that the Trp was located inside the lipid bilayer. The quenching indicated that I- binds to positive charges of the protein located on the surface of the membrane. This, combined with knowledge of the sequence of lipophilin, suggested that Trp-211 was located within the membrane but was close to amino acid residues that are external to the bilayer.     

Conformation of amyloid fibrils of beta2-microglobulin probed by tryptophan mutagenesis

Beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, adopts an immunoglobulin domain fold in its native state. Although beta2-m has Trp residues at positions 60 and 95, both are located near the surface of the domain. Hence, beta2-m does not have a conserved Trp common to other immunoglobulin domains, which is buried in close proximity to the disulfide bond. To study the structure of amyloid fibrils in relation to their native fold, we prepared a series of Trp mutants. Trp60 and Trp95 were both replaced with Phe, and a single Trp was introduced at various positions. Among various mutants, W39-beta2-m, in which a Trp was introduced at the position corresponding to the conserved Trp, exhibited a remarkable quenching of fluorescence in the native state, as observed for other immunoglobulin domains. An x-ray structural analysis revealed that W39-beta2-m assumes the native fold with Trp39 located in the vicinity of the disulfide bond. Comparison of the fluorescence spectra of various mutants for the native and fibrillar forms indicated that, while the Trp residues introduced in the middle of the beta2-m sequence tend to be buried in the fibrils, those located in the C-terminal region are more exposed. In addition, the fluorescence spectra of fibrils prepared at pH 2.5 and 7.0 revealed a large difference in the fluorescence intensity for W60-beta2-m, implying a major structural difference between them.     

Rapid kinetics of Na+ binding to thrombin

The kinetic mechanism of Na(+) binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na(+) binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k(obs) decreases hyperbolically with increasing [Na(+)]. The rapid phase is due to Na(+) binding to the enzyme E to generate the E:Na(+) form. The slow phase is due to the interconversion between E(*) and E, where E(*) is a form that cannot bind Na(+). Temperature studies in the range from 5 to 35 degrees C show significant enthalpy, entropy, and heat capacity changes associated with both Na(+) binding and the E to E(*) transition. As a result, under conditions of physiologic temperature and salt concentrations, the E(*) form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na(+) (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na(+) binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na(+) binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.     

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.     

Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2

Highly purified plasminogen-activator inhibitors of type 1 (PAI-1) and type 2 (PAI-2), low-Mr form, were compared with respect to their kinetics of inhibition of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). The time course of inhibition of plasminogen activator was studied under second-order or pseudo-first-order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t-PA or u-PA substrate or by an immunosorbent assay for t-PA activity. PAI-1 rapidly reacted with single-chain t-PA as well as with two-chain forms of t-PA and u-PA. The second-order rate constant k for inhibition of single-chain t-PA (5.5 x 10(6) M-1 s-1) was about three times lower than k for inhibition of the two-chain activators. PAI-2 reacted slowly with single-chain t-PA, k = 4.6 x 10(3) M-1 s-1. The association rate was 26 times higher with two-chain t-PA and 435 times higher with two-chain u-PA. The k values for inhibition of single-chain t-PA, two-chain t-PA and two-chain u-PA were respectively, 1200, 150 and 8.5 times higher with PAI-1 than with PAI-2. The removal of the epidermal growth factor domain and the kringle domain from two-chain u-PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C-terminal proteinase part of u-PA (B chain) is responsible for both the primary and the secondary interactions with PAI-1 and PAI-2. The k values for inhibition of single-chain t-PA and endogenous t-PA in plasma by PAI-1 or PAI-2 were identical indicating that t-PA in blood consists mainly in its single-chain form.     

Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein

SecA ATPase is an essential component of the Sec-dependent protein translocation machinery. Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane. To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions. Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure. Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins. Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands. Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide. These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.