Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.     

Ovomucoid domains: preparation and physico-chemical characterization

Four fragments of ovomucoid representing its individual domains and their different combinations were prepared by peptic and cyanogen bromide cleavages of the protein. The fragments corresponding to domains I + II, II + III, I and III of the parent ovomucoid molecule, were found to be homogeneous by gel filtration and polyacrylamide gel electrophoresis in presence and absence of SDS. Various physico-chemical properties of these proteins, such as molecular weight, NH2- and COOH-terminal amino acid residues, sugar content, isoionic pH, specific extinction coefficient, fluorescence emission spectra, intrinsic viscosity, frictional coefficient, Stokes radius, diffusion coefficient and geometrical mean radius were determined. Analysis of the results on trypsin inhibitory activity of ovomucoid and its different fragments suggested that only domain II is involved in the antitryptic activity of the inhibitor. Optical characteristics of these fragments indicate that they are devoid of tryptophan residues. The hydrodynamic properties suggest that intact ovomucoid and two of its fragments (domain I + II and domain II + III) are significantly different from those of typical globular proteins and are asymmetric in nature. However, the shape of the two remaining fragments representing domains I and III of the intact protein appeared to be compact and globular. Furthermore, domain II of ovomucoid has been suggested to primarily contribute towards the apparent asymmetry in the intact protein.     

Chemical deglycosylation of hen ovomucoid: protective effect of carbohydrate moiety on tryptic hydrolysis and heat denaturation

Hen ovomucoid was chemically deglycosylated by treatment with trifluoromethanesulfonic acid at 0 degrees C for 60 min. About 75 mol% of the carbohydrate moiety was removed from the glycoprotein without changing its amino acid composition, and its trypsin inhibitory activity and immunoreactivity with specific antibodies remained unchanged. The deglycosylated ovomucoid was inactivated and degraded easily by an excess amount of trypsin, whereas the native glycoprotein was not. Furthermore, the biological and immunological activities of the deglycosylated ovomucoid were lowered by heat treatment more easily than those of the native ovomucoid. These results suggest that the carbohydrate moiety of ovomucoid contributes to the stability of the ovomucoid molecule against tryptic hydrolysis and heat denaturation.     

Differential scanning calorimetric,circular dichroism,and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.     

tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX.

The tau and gamma proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with gamma being a truncated version of tau arising from ribosomal frameshifting. tau is comprised of five structural domains, the first three of which are shared by gamma (Gao, D., and McHenry, C. (2001) J. Biol. Chem. 276, 4433-4453). In the absence of the other holoenzyme subunits, DnaX exists as a tetramer. Association of delta, delta', chi, and psi with domain III of DnaX(4) results in a DnaX complex with a stoichiometry of DnaX(3)deltadelta'chipsi. To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V. Unlike domain I-II, treatment of domain III-V, gamma, and tau with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein. Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer. Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting tau, delta, delta', chi, and psi onto streptavidin-agarose beads. Thus, domain III not only contains the delta, delta', chi, and psi binding interface, but also the region that enables DnaX to oligomerize.     

Immunochemical studies on thermal denaturation of ovomucoid

The thermal denaturation of ovomucoid was investigated by immunochemical methods, namely immunoprecipitation analyses and antibody-Sepharose 4B column chromatography. In the immunoprecipitation analyses, heated ovomucoid (90 degrees C, 90 min, pH 7.2) required about twice the antigen addition of the native protein to approach maximal precipitation with specific antibody, and the maximal immunoprecipitation was decreased to 80% of that by native ovomucoid. However, heated protein inhibited the binding of antibody with native ovomucoid, and 100% inhibition was attained at about 4-times the antigen addition necessary for the native protein. Heated ovomucoid (100 degrees C, 120 min) showed little immunoprecipitation and inhibition. To ovomucoid antigenicity was diminished more slowly than the trypsin inhibitory activity by heating, e.g., heated ovomucoid (90 degrees C, 120 min) retained more than 30% of the antigenicity but little trypsin inhibitory activity. By passing through the immunoaffinity column, heated ovomucoid (90 degrees C, 90 min) was separated into two fractions, either with (fraction II) or without (fraction I) antigenicity. Fraction II contained smaller fractions of ordered secondary structure than native ovomucoid, and trypsin inhibitory activity of fraction II was only 24% of the native one. These results indicated that thermally denatured ovomucoid was heterogeneous regarding the conformational damage caused by heating, and the structure around some antigenic sites in an ovomucoid molecule was retained even after the backbone conformation was partially destroyed and trypsin inhibitory activity was lost.     

Different extracellular domains of the neural cell adhesion molecule (N- CAM) are involved in different functions

The neural cell adhesion molecule (N-CAM) engages in diverse functional roles in neural cell interactions. Its extracellular part consists of five Ig-like domains and two fibronectin type III homologous (type III) repeats. To investigate the functional properties of the different structural domains of the molecule in cell interactions and signal transduction to the cell interior, we have synthesized, in a bacterial expression system, the individual domains and tandem sets of individual domains as protein fragments. These protein fragments were tested for their capacity to influence adhesion and spreading of neuronal cell bodies, promote neurite outgrowth, and influence cellular migration patterns from cerebellar microexplants in vitro. Ig-like domains I and II and the combined type III repeats I-II were most efficient for adhesion of neuronal cell bodies, when coated as substrates. Neurite outgrowth was best on the substrate-coated combined type III repeats I- II, followed by the combined Ig-like domains I-V and Ig-like domain I. Spreading of neuronal cell bodies was best on substrate-coated combined type III repeats I-II, followed by Ig-like domain I and the combined Ig- like domains I-V. The cellular migration pattern from cerebellar microexplant cultures plated on a mixture of laminin and poly-L-lysine was modified by Ig-like domains I, III, and IV, while Ig-like domains II and V and the combined type III repeats I-II did not show significant modifications, when added as soluble fragments. Outgrowth of astrocytic processes from the explant core was influenced only by Ig- like domain I. Metabolism of inositol phosphates was strongly increased by Ig-like domain I and less by the Ig-like domains II, III, IV, and V, and not influenced by the combined type III repeats I-II. Intracellular concentrations of Ca2+ and pH values were increased only by the Ig-like domains I and II. Intracellular levels of cAMP and GMP were not influenced by any protein fragment. These experiments indicate that different domains of N-CAM subserve different functional roles in cell recognition and signal transduction, and are functionally competent without nervous system-derived carbohydrate structures.     

Two beta-rich structural domains in GABA(A) receptor alpha(1) subunit with different physical properties: Evidence for multidomain nature of the receptor

The type A gamma-aminobutyric acid (GABA(A)) receptor is a major inhibitory neurotransmitter-gated ion channel. Previously, we identified a membrane-proximal beta-rich (MPBR) domain in fragment C166-L296 of GABA(A) receptor alpha(1) subunit, forming nativelike pentamers. In the present study, another structural domain, the amino-terminal domain, was shown to exist in the fragment Q28-E165. The secondary structures of both fragments were beta-rich as measured using FTIR spectroscopy and estimated from the CD spectra to be 42% and 51% beta-strand for Q28-E165 and C166-L296, respectively. The CD spectrum of the combined fragment Q28-L296 was additive of the spectra of the two fragments. In addition, denaturation curves of both fragments were characteristic of cooperative transitions, supporting their domainlike nature. C166-L296 required 6.5 M of guanidine chloride for total denaturation, therefore it is extraordinarily stable, more so than Q28-E165. Moreover, effects of detergent on the molecular masses of Q28-E165 and C166-L296, as monitored using laser-scattering spectroscopy, indicated that intermolecular interactions were much more significant in C166-L296 than in Q28-E165. Effects of pH on their molecular masses suggested that ionic forces were involved in these interactions. Together the results show that the two adjacent fragments form independent folding units, MPBR and amino-terminal domains, different in secondary structure content, denaturation profile, and polymerization status, and suggest that the former may play a more important role in receptor assembly and that the extraordinary stability may underlie its intrinsic tendency to form oligomers. More significantly, the present study has provided direct evidence for the long-postulated multidomain nature of this family of receptors.     

Two structural domains as a general fold of the toxic fragment of the Bacillus thuringiensis delta-endotoxins.

The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two-step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265, 1369-1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease-susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N-terminal half of both toxins.     

C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains

C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human histidine-rich glycoprotein: simultaneous purification with antithrombin III and characterization of its gross structure

The simple and simultaneous purification of histidine-rich glycoprotein (HRG) and antithrombin III (AT III) from human plasma and gross structural characterization of HRG have been performed. The purification method consists of two chromatographic procedures using heparin-agarose and DEAE-Sephadex. The yields of HRG and AT III were 22 mg and 70 mg, respectively, from 1 liter of plasma. The purified HRG is a single-chain polypeptide with a molecular weight (Mr) of 75,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, indicating it was the native form of this protein. Cyanogen bromide cleavage of HRG, followed by analysis of the amino acid composition and determination of the amino-terminal sequence of each purified cyanogen bromide fragment established that the gross structure of HRG consisted of three cyanogen bromide fragments; an amino-terminal CN-50 kDa fragment (Mr 50,000) and a carboxy-terminal small fragment of eight amino acids, and a CN-30 kDa fragment (Mr 30,000) between them. As to the amino acid composition of the CN-30 kDa fragment, it had an unusually high content of histidine (25 mol%), suggesting the presence of a histidine-rich region(s) in the carboxy-terminal half of the molecule. These results together with our previous results (Koide, T., Odani, S., & Ono, T. (1982) FEBS Lett. 141, 222-224) and those of Morgan (Morgan, W.T. (1985) Biochemistry 24, 1496-1501) imply that HRG is composed of at least two domains with distinct functional properties; i.e. an amino-terminal domain with heparin-binding ability and a carboxy-terminal domain with heme- and divalent metal-binding abilities.     

Conformation and stability of the anion transport protein of human erythrocyte membranes

The conformation and stability of purified preparations of band 3, the anion transport protein of human erythrocyte membranes, and its constituent proteolytic subfragments have been studied by circular dichroism. Band 3, purified in the presence of the nonionic detergent n-dodecyl octaethylene glycol monoether (C12E8), had an alpha-helical content of 46%. Denaturation of purified band 3 with guanidine hydrochloride occurred in two phases, one reflecting much more resistance to denaturation than the other. Band 3 can be separated into two domains by limited in situ proteolytic cleavage. The carboxyl-terminal membrane-associated domain (Mr 55 000) purified in C12E8 contained 58% alpha-helix and was very resistant to denaturation by guanidine hydrochloride. The purified amino-terminal, cytoplasmic domain (Mr 41 000) contained 27% alpha-helix and was completely converted to a random-coil conformation by 3 M guanidine hydrochloride. The two phases of denaturation observed for intact band 3 corresponded to the two domains of the protein. Irreversible heat denaturation of purified band 3 occurred with half-maximal change in theta 222.5 at 48 degrees C. Covalent attachment of the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate to band 3 had little effect on the circular dichroism spectra of band 3 or the membrane-associated domain but resulted in stabilization of band 3 to heat denaturation (half-maximal change in theta 222.5 = 61 degrees C). Circular dichroism studies of membranes that had been digested extensively with proteolytic enzymes and stripped of all extrinsic fragments revealed that the portions of red cell membrane proteins that are embedded in the lipid bilayer contain a very high (86-94%) content of alpha-helix.     

The scFv fragment of the antibody hu4D5-8: evidence for early premature domain interaction in refolding

Fluorescence spectroscopy and 1H/2H-exchange techniques have been applied to characterize the folding of an scFv fragment, derived from the humanized anti-HER2 antibody hu4D5-8. A stable intermediate, consisting of a native VL domain and an unfolded VH domain, is populated under equilibrium unfolding conditions. A partially structured intermediate, with 1H/2H-exchange protection significantly less than that of the two isolated domains together, is detectable upon refolding the equilibrium-denatured scFv fragment. This means that the domains in the heterodimer do not fold independently. Rather, they associate prematurely before full 1H/2H-exchange protection can be gained. The formation of the native heterodimer from the non-native intermediate is a slow, cooperative process, which is rate-limited by proline cis/trans-isomerization. Unproductive domain association is also detectable after short-term denaturation, i.e. with the proline residues in native conformation. Only a fraction of the short-term denatured protein folds into the native protein in a fast, proline-independent reaction, because of spontaneous proline cis/trans-reisomerization in the early non-native intermediate. The comparison with the previously studied antibody McPC603 has now allowed us to delineate similarities in the refolding pathway of scFv fragments.     

Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway

The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.     

Analysis of the interactions between streptokinase domains and human plasminogen.

The contrasting roles of streptokinase (SK) domains in binding human Glu1-plasminogen (Plg) have been studied using a set of proteolytic fragments, each of which encompasses one or more of SK's three structural domains (A, B, C). Direct binding experiments have been performed using gel filtration chromatography and surface plasmon resonance. The latter technique has allowed estimation of association and dissociation rate constants for interactions between Plg and intact SK or SK fragments. Each of the SK fragments that contains domain B (fragments A2-B-C, A2-B, B-C, and B) binds Plg with similar affinity, at a level approximately 100- to 1,000-fold lower than intact SK. Experiments using 10 mM 6-aminohexanoic acid or 50 mM benzamidine demonstrate that either of these two lysine analogues abolishes interaction of domain B with Plg. Isolated domain C does not show detectable binding to Plg. Moreover, the additional presence of domain C within other SK fragments (B-C and A2-B-C) does not alter significantly their affinities for Plg. In addition, Plg-binding by a noncovalent complex of two SK fragments that contains domains A and B is similar to that of domain B. By contrast, species containing domain B and both domains A and C (intact SK and the two-chain complex A1 x A2-B-C) show a significantly higher affinity for Plg, which could not be completely inhibited by saturating amounts of 6-AHA. These results show that SK domain B interacts with Plg in a lysine-dependent manner and that although domains A and C do not appear independently to possess affinity for Plg, they function cooperatively to establish the additional interactions with Plg to form an efficient native-like Plg activator complex.     

Molecular dynamics simulation of Escherichia coli dihydrofolate reductase and its protein fragments: relative stabilities in experiment and simulations

We have carried out molecular dynamics simulations of the native dihydrofolate reductase from Escherichia coli and several of its folded protein fragments at standard temperature. The simulations have shown fragments 1--36, 37--88, and 89--159 to be unstable, with a C(alpha)RMSD (C(alpha) root mean squared deviation) >5 A after 3.0 nsec of simulation. The unfolding of fragment 1--36 was immediate, whereas fragments 37--88 and 89--159 gradually unfolded because of the presence of the beta-sheet core structure. In the absence of residues 1--36, the two distinct domains comprising fragment 39--159 associated with each other, resulting in a stable conformation. This conformation retained most of its native structural elements. We have further simulated fragments derived from computational protein cutting. These were also found to be unstable, with the exception of fragment 104--159. In the absence of alpha(4), the loose loop region of residues 120--127 exhibited a beta-strand-like behavior, associating itself with the beta-sheet core of the protein fragment. The current study suggests that the folding of dihydrofolate reductase involves cooperative folding of distinct domains which otherwise would have been unstable as independent folded units in solution. Finally, the critical role of residues 1--36 in allowing the two distinct domains of fragment 104--159 to fold into the final native conformation is discussed.     

Structural stability and domain organization of colicin E1

Thermodynamic properties, stability, and structure of the toxin-like molecule colicin E1 were analyzed by differential scanning calorimetry and circular dichroism to determine the number of structurally independent domains, and the interdomain interactions necessary for colicin import into the Escherichia coli cell. Analysis of denaturation profiles of the 522 residue colicin E1, together with fragments of 342 and 178 residues that contain subsets of the domains, showed three stable cooperative blocks that differ in thermal stability and correspond to three major functional domains of the colicin: (i) the COOH-terminal channel-forming (C) domain with the highest thermal stability; (ii) the BtuB receptor binding (R) domain; and (iii) the N-terminal translocation (T) domain that has the smallest stabilization enthalpy and thermal stability. Interdomain interactions were described in which T-R interactions stabilize R, and T-C and R-C interactions stabilize R and T, but destabilize C. The R and T domains behaved in a similar way as a function of pH and ionic strength. Interacting extended helices of the R domain, possibly a coiled-coil, were implied by: (i) the very high (>90%) alpha-helical content of the R domain, (ii) cooperative decreases in alpha-helical content near the T(tr) of thermal denaturation of the R domain; (iii) a large denaturation enthalpy, implying extensive H-bond and van der Waals interactions. The R domain was inferred, from the extended network of interacting helices, large DeltaH, and steep temperature dependence of its stabilization energy to have a dominant role in determining the conformation of other domains. It is proposed that cellular import starts with the R domain binding to the BtuB receptor, followed by unfolding of the R domain coiled-coil and thereby of the T domain, which then interacts with the TolC receptor-translocator.     

Chicken ovomucoid: determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains

The complete amino acid sequence of chicken ovomucoid (OMCHI) is presented. OMCHI consists of three tandem domains, each homologous to pancreatic secretory trypsin inhibitor (Kazal) and each with an actual or putative reactive site for inhibition of serine proteinases. The major reactive site for bovine beta-trypsin is the Arg89-Ala peptide bond in the second domain. The equilibrium constant for hydrolysis of this peptide bond, K0hyd, is 1.85. The first and third domains of OMCHI are relatively ineffective inhibitors of several serine proteinases against which they were tested. OMCHI is a mixture of two forms: the major form with all of the amino acid residues and a minor form with Val134-Ser135 deleted. This polymorphism is present in all chicken eggs and is the result of ambiguous excision at the 5' end of the F intron. Procedures are given for preparation of modified chicken ovomucoid, OMCHI (in which the Arg89-Ala bond is hydrolyzed), of the first domain, OMCHI1 (residues 1-68), of the second domain, OMCHI2 (residues 65-130), and of the third domain, OMCHI3 (residues 131-186). In the case of the third domain, both the Asn175 glycosylated form, OMCHI3(+), and the carbohydrate-free form, OMCHI3(-), were obtained. These isolated native domains are useful in many studies of ovomucoid behavior.     

The primary structure of a new Mr 18,000 calcium vector protein from amphioxus

The amino acid sequence of a new Ca2+-binding protein (CaVP) from Amphioxus muscle (Cox, J. A., J. Biol. Chem. 261, 13173-13178) has been determined. The protein contains 161 amino acid residues and has a molecular weight of 18,267. The N terminus is blocked by an acetyl group. The two functional Ca2+-binding sites have been localized based on homology with known Ca2+-binding domains, on internal homology and on secondary structure prediction, and appear to be the domains III and IV. The C-terminal half of CaVP, which contains the two Ca2+-binding sites, shows a remarkable similarity with human brain calmodulin (45%) and with rabbit skeletal troponin C (40%). Functional domain III contains 2 epsilon-N-trimethyllysine residues in the alpha-helices flanking the Ca2+-binding loop. Sequence determination revealed two abortive Ca2+-binding domains in the N-terminal half of CaVP with a similarity of 24 and 30% as compared with calmodulin and troponin C, respectively. This half is also characterized by the presence of a disulfide bridge linking the N-terminal helix of domain I to the C-terminal helix of domain II. This disulfide bond is very resistant to reduction in the native state, but not in denatured CaVP. The optically interesting aromatic chromophores (2 tryptophan and 1 tyrosine residues) are all located in the nonfunctional domain II.     

Transition state of the rate-limiting step of heat denaturation of Cry3A delta-endotoxin

Heat denaturation of Cry3A delta-endotoxin from Bacillus thuringiensis var. tenebrionis and its 55 kDa fragment was studied by differential scanning microcalorimetry at low pH. Analysis of the calorimetric data has shown that denaturation of Cry3A delta-endotoxin is a nonequilibrium process at heating rates from 0. 125 to 2 K/min. This means that the stability of delta-endotoxin (the apparent temperature of denaturation Tm) under these conditions is under kinetic control rather than under thermodynamic control. It has been shown that heat denaturation of this protein is a one-step kinetic process. The enthalpy of the process and its activation energy were measured as functions of temperature. The data obtained allow confirmation of the fact that the conformation of delta-endotoxin at the transition state only slightly differs from its native conformation with respect to compactness and extent of hydration. The comparison of the activation energy for intact delta-endotoxin and the 55 kDa fragment showed that the transition of the molecule to a transition state does not cause any changes in the conformation of three N-terminal alpha-helices. Complete removal of the N-terminal domain of delta-endotoxin and 40 amino acids from the C-terminus beta-sheet domain III causes an irreversible loss of the tertiary structure. Thus, during protein folding the nucleation core determining protein stability does not involve its three initial alpha-helices but may include the remaining alpha-helices of the N-terminal domain. The functional significance of peculiarities of structure arrangement of the delta-endotoxin molecule is discussed.