Chemical and functional characterization of a fragment of C1-s containing the epidermal growth factor homology region

C1-s, one of the three subcomponents of C1-, the first component of complement, is a serine protease comprising two disulfide-linked chains, the B chain, containing the catalytic site, and the A chain, involved in Ca2+ binding and Ca2(+)-dependent interaction(s) with the other C1- subcomponents. In an attempt to identify the regions responsible for the latter functions, C1-s was submitted to limited proteolysis with plasmin, a treatment that split the A chain into three major fragments, alpha 1, alpha 2, and gamma. Fragment alpha 2, which comprised the epidermal growth factor-like (EGF-like) region of C1-s, was heterogeneous, starting at serine 97 or phenylalanine 105 and ending at lysine 195. This fragment was reduced and alkylated and then digested with elastase, and three peptides covering positions 131-135, 131-139, and 131-140 were characterized by amino acid analysis, Edman degradation, and mass spectrometry, showing that position 134 of C1-s is occupied partly by an asparagine (47%) and partly by an erythro-beta-hydroxyasparagine, in contrast with the homologous position (150) of C1-r which only contains erythro-beta-hydroxyasparagine. As measured by equilibrium dialysis, native alpha 2, like the other plasmin-cleavage fragments, did not retain the ability of intact C1-s to bind Ca2+. In the same way, plasmin cleavage abolished the ability of C1-s to dimerize or to associate with C1-r in the presence of Ca2+. In contrast, both alpha 2 and the N-terminal alpha 1 fragment, starting at serine 24 of the A chain, were able to compete significantly with intact C1s for the formation of the Ca2(+)-dependent C1-s-C1r-C1-r-C1-s tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r

Fluorescent probes and other methods have been used to investigate the thermal stability of activated C1r and functionally intact fragments isolated from tryptic digests of the protein. This enzyme exhibits two irreversible transitions that differ with respect to their sensitivity to metal ions. The high-temperature transition occurs with a midpoint near 53 degrees C in 0.02 M tris(hydroxymethyl)aminomethane buffer and 0.15 M NaCl, pH 7.4. It is relatively insensitive to Ca2+ and ionic strength and is accompanied by a loss of catalytic activity. The low-temperature transition is most easily observed in the presence of ethylenediaminetetraacetic acid and is completely abolished by 100 microM Ca2+. Its midpoint varies between 26 degrees C at low ionic strength and 40 degrees C in the presence of 0.5 M NaCl. The low-temperature transition results in extensive polymerization of the protein without loss of the esterolytic activity or the ability to react with C1 inhibitor; however, the ability to reconstitute hemolytically active C1 or even bind to C1s in the presence of Ca2+ is destroyed. A highly purified N-terminal fragment generated by tryptic digestion of C1r in the presence of Ca2+ retained its ability to interact with C1s, disrupting the formation of C1s dimers in the presence of Ca2+. In the absence of Ca2+, this fragment displays only a low-temperature transition that is very similar to the one observed with the whole protein and that destroys its ability to bind to C1s. Addition of Ca2+ stabilizes this fragment, shifting the midpoint of its melting transition upward by more than 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins

Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.     

Molecular characterization of the catalytic domains of human complement serine protease C1r

Limited cleavages of human C1r by extrinsic proteases of various specificity (plasmin, elastase, chymotrypsin, thermolysin) yield dimeric associations of two globular domains, each comprised of the intact B chain disulfide linked to gamma, the C-terminal fragment of the A chain. These (gamma-B)2 domains, which are homologous to those obtained from C1r by autolytic cleavage [Villiers, C. L., Arlaud, G. J., & Colomb, M. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4477-4481], represent the core of the C1r molecule and are associated with the catalytic properties of the serine active site. V8 protease also yields (gamma-B)2 associations, although additional cleavages occur in the B chain. Sequence analysis shows that all cleavages generating the gamma fragments occur within a 13-residue sequence extending from positions 274 to 286 of the C1r A chain. Chemical cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide of the (gamma-B)2 catalytic domains obtained from C1r autolytic cleavage indicates that each gamma-B domain interacts with its neighbor in a "head to tail" configuration, the gamma region of one domain interacting with the B chain of the other domain, and conversely. No evidence is found of gamma-gamma or B-B interactions. Such a head to tail configuration, placed in the context of the model proposed for the C1s-C1r-C1r-C1s catalytic subunit of C1 [Colomb, M. G., Arlaud, G. J., & Villiers, C. L. (1984) Philos. Trans. R. Soc. London, B 306, 283-292], is compatible with autolytic activation of C1r through an intramolecular cross-mechanism and with subsequent activation of C1s by activated C1r.     

Specific DNA binding of the cAMP receptor protein within the lac operon stabilizes double-stranded DNA in the presence of cAMP

The effects of varying amounts of cAMP receptor protein (CRP) in the presence and absence of cAMP on the melting and differential melting curves of a 301-bp fragment containing the lac control region in 5 mM Na+ have been investigated. The native 301-bp fragment consists of three cooperatively melting thermalites. At 5 mM Na+, thermalite I (155 bp) has a Tm of 66.4 degrees C and the melting transitions of thermalites II (81 bp) and III (65 bp) are superimposed with a Tm of 61.9 degrees C. The specific DNA target site for CRP and the lac promotor are located within thermalite II. CRP alone exerts no specific effects on the melting of the 301-bp fragment, non-specific DNA binding of CRP resulting in a progressive stabilization of the double-stranded DNA by increasing the number of base pairs melting at a higher Tm in a non-cooperative transition. The cAMP-CRP complex, however, exerts a specific effect with a region of approximately 36 bp, comprising the specific CRP binding site and a neighbouring region of DNA, being stabilized. The appearance of this new cooperatively melting region, known as thermalite IV, is associated with a corresponding decrease in the area of thermalites II/III. The Tm of thermalite IV is 64.4 degrees C, 2.5 degrees C higher than that of thermalites II/III. With two or more cAMP-CRP complexes bound per 301-bp fragment, the stabilization also affects the remaining 110 bp now making up thermalites II/III whose Tm is increased by 1 degrees C to 62.9 degrees C. The implications of these findings for various models of the mode of action of the cAMP-CRP complex are discussed.     

Relative biological effectiveness of the alpha-particle emitter (211)At for double-strand break induction in human fibroblasts

The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the alpha-particle emitter (211)At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with alpha particles from (211)At, (60)Co gamma rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp-5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with alpha particles from (211)At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for (60)Co gamma rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with (211)At alpha particles or (60)Co gamma rays at 2 degrees C or 37 degrees C and intact cells were irradiated with (211)At alpha particles at the same temperatures. The dose-modifying factor (DMF(temp)) for irradiation of nuclear monolayers at 37 degrees C compared with 2 degrees C was 1.7 for (211)At alpha particles and 1.6 for (60)Co gamma rays. No temperature effect was observed for intact cells irradiated with (211)At. In conclusion, irradiation with alpha particles from (211)At induced two to three times more DSB than gamma rays and X rays.     

Immunochemical determination of conformational equilibria for fragments of the B beta chain of fibrinogen

The conformations of the B beta chain of the intact fibrinogen molecule and of various fragments of the B beta chain of fibrinogen that contain the region that is hydrolyzed by thrombin have been compared by an immunochemical method [Sachs, D. H., Schechter, A. N., Eastlake, A., & Anfinsen, C. B. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3790]. Anti-fibrinogen antibodies were induced in rabbits by immunization with native bovine fibrinogen. An antibody population specific for the native antigenic determinant within the B beta fragment 20-28 was isolated by immunoadsorption. This preparation was to determine the value of Kconf, the equilibrium constant for the interconversion of the nonnative and native conformations of this determinant. Values of Kconf were measured for this determinant within native fibrinogen, the disulfide knot (DSK), CNBrB beta, B beta fragment 16-28, B beta fragment 20-28, and fibrinopeptide B (FpB). 125I-Labeled fibrinogen (125I-F) was used in the determination of Kconf by measuring the competition between 125I-F and the fibrinogen derivatives under study for binding to the purified antibody. For the antigenic region in F, the DSK, and CNBrB beta, the values of Kconf at 4 degrees C were infinity, (5.9 +/- 3.5) X 10(-3), and (1.2 +/- 0.7) X 10(-3), respectively. The values of Kconf for B beta fragment 16-28, B beta fragment 20-28, and FpB at 4 degrees C were less than (6.0 +/- 3.9) X 10(-7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of chain unsaturation on the structure and thermotropic properties of galactocerebrosides.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the effect of increasing chain-unsaturation on the structure and properties of the hydrated cerebrosides N-stearoyl, -oleoyl, and -linoleoyl galactosylsphingosine (NSGS, NOGS, and NLnGS, respectively). DSC of hydrated (70 wt% water) NSGS shows an endothermic transition at 85 degrees C (delta H = 18.0 kcal/mol NSGS) and a broad exothermic transition at 40-60 degrees C, the latter being dependent upon the previous cooling rate. X-Ray diffraction patterns recorded at 21, 61, and 86 degrees C provide evidence for interconversions between metastable and stable crystalline NSGS bilayer phases. The properties of the unsaturated-chain cerebrosides are more complex. Hydrated NOGS shows a single endothermic transition at 44.8 degrees C (delta H = 11.5 kcal/mol NOGS). However, incubation of NOGS at 49 degrees C for 24 h results in a second transition at 55.5 degrees C. By cycling NOGS between 0 and 49 degrees C complete conversion into this higher melting phase (delta H = 12.1 kcal/mol NOGS) is achieved. X-ray diffraction confirms a bilayer phase at all temperatures and delineates the conversions between a crystalline phase at 21 degrees C (bilayer period d = 56.5A), a second crystalline phase at 47 degrees C (d = 69.9A), and a liquid crystalline phase at 59 degrees C (d = 52.0A). The more unsaturated NLnGS shows two transitions, a sharp transition at 28 degrees C (delta H = 8.0 kcal/mol NLGS) and a broad, low-enthalpy transition at 42 degrees C (delta H = 0.4 kcal/mol NLGS). Again, incubation between the two transitions leads to a single transition at 44 degrees C (delta H = 9.3 kcal/mol NLGS). X-ray diffraction demonstrates conversions between two crystalline bilayer phases (d = 55.2A and d = 68.4A), and a liquid crystalline bilayer phase (d = 51.8A). Thus, increased unsaturation in the amide-linked fatty acyl chain of cerebrosides results in decreased chain-melting temperatures (NSGS greater than NOGS greater than NLnGS) and has marked effects on their structural properties.     

Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E

In order to study thrombin interaction with fibrinogen, thrombin binding to fragments D and E (prepared by plasmin digestion of fibrinogen) and to intact S-carboxymethylated chains of fibrinogen (A alpha, B beta, and gamma) was analyzed by autoradiography, immunoblotting, and affinity chromatography. Complex formation was observed between late fragment E and thrombin but not with fragment D. The three reduced chain remnants of fragment E all formed complexes with thrombin. Also, thrombin bound to the intact, separated A alpha, B beta, and gamma chains of fibrinogen as well as to the alpha and beta chains of fibrin. In these experiments the extended substrate-binding site, but not the catalytic-binding site, was being examined because fragment E had as its amino-terminal amino acids Val20 in the alpha chain, Lys54 in the beta chain, and Tyr1 in the gamma chain. Also, thrombin inhibited in its active center by D-phenyl-alanyl-L-prolyl-L-arginine-chloromethyl ketone bound to fragment E and to the separated chains in the same manner as unmodified thrombin. A lysine residue to thrombin was essential for its binding to fibrinogen. Thrombin attached to CNBr-activated Sepharose through its amino groups did not bind to fragment E, but when thrombin was attached through its carboxyl groups, it bound fragment E.     

Correlation of thermodynamic and genetic properties in the Tn10 encoded TET gene control region.

The thermal stability of the Tn10 encoded tetracycline resistance (TET) gene control region is investigated by melting studies using purified DNA restriction fragments containing various amounts of flanking sequences. In order to study the thermodynamic properties of this control region under conditions, where enough flanking DNA is present to mimic the situation in the chromosome, the five step melting process of a 1450-bp DNA fragment is analyzed. Because most of the sequence of this DNA is not known, the assignment of the melting transitions to segments of the DNA is done by an experimental method. This employs the preparation of subfragments from the 1450-bp DNA and comparison of their denaturation profiles with the one of the intact sequence. This approach results in the complete assignment of the five denaturation steps. Rather than from the ends, the unwinding starts from the TET gene control region in the middle of the 1450-bp sequence. A clear correlation between the thermodynamic and genetic properties of this DNA is observed. The regulatory sequence forms a small cooperative unit with the lowest stability in the entire fragment. The thermal denaturation of the TET repressor. TET operator complex reveals, that the TET repressor specifically recognizes the double stranded TET operator DNA and stabilizes this structure by 2.4 degrees C. This results is also discussed as an example of the possible action of denaturing or stabilizing proteins on this genetic control region.     

The presence of cooperative structures in tryptic fragments of troponin C

Scanning microcalorimetry has been used to study the ability of TR-1 and TR-2 tryptic fragments of troponin C to form an ordered compact structure in solution under different conditions. It has been shown that: (1) in the presence, as well as in the absence of bivalent ions both fragments have a structure which can melt with an intensive heat absorption at heating; (2) the structure of fragment TR-1 containing two Ca2+-specific domains (domains I and II) melts as a whole under all conditions studied and therefore the domains form one cooperative block. Binding of Ca2+ or Mg2+ ions stabilizes the block structure, however, significant conformational rearrangements which would lead to a change of denaturational enthalpy do not occur; (3) Ca2+Mg2+-domains of fragment TR-2 (domains III and IV) represent individual cooperative units, blocks. Stability of these cooperative blocks strongly depends on concentration of bivalent ions and in the presence of 2 mM EDTA the melting temperature of one of them is below 10 degrees. Thermodynamic melting temperature of one of them is below 10 degrees. Thermodynamic melting parameters of cooperative blocks within peptides and in the intact molecule of troponic C are compared.     

The 3'' terminus of 16S rRNA: secondary structure and interaction with ribosomal protein S1.

We report studies of the secondary structure and S1 ribosomal protein binding properties of the colicin fragment, containing 49 residues from the 3' terminus of E. coli 16S rRNA. Temperature jump relaxation kinetic measurements reveal two helices in the structure. One of these, melting at 81 degrees C in 5 mM Mg2+, is associated with the 9-base pair hairpin helix predicted by the nucleotide sequence. The other melting transition, at 21 degrees C in 5 mM Mg2+, is assigned to a 4-base pair helix which constrains the pyrimidine tract of the colicin fragment into a bulge loop. S1 protein forms a strong 1:1 complex with the colicin fragment, with an association constant of 5 x 10(6) M-1 in 5 mM Mg2+. More protein molecules are bound, but with weaker affinity, when the S1 concentration is increased. S1 binding causes melting of the colicin fragment secondary structure, as inferred from the observed absorbance increase. The S1 binding site on the colicin fragment has been localized in the region of the bulge loop, since the melting transition corresponding to the 4-base pair helix is lost in the complex. We discuss current models for the role of S1 protein in polypeptide chain initiation in light of these and previous results.     

Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a plasmin-digest fragment of rabbit immunoglobulin gamma that binds antigen and complement.

Rabbit immunoglobulin gamma (IgG) was digested with plasmin after being left for 15 min at pH2.5, 30 degrees C followed by a rapid increase in the pH to 7. The fragment antigen and complement binding (Facb) was isolated and characterized chemically and biologically. Sequence studies showed that the C-terminal quarter of the heavy chain had been removed, the split occurring at a lysine-alanine bond in the sequence Thr-Ile-Ser-Lys-Ala-Arg. The fragment Facb retained the capacity to precipitate with antigen and the precipitate caused activation of the first component of complement of the same order as that of acid-treated IgG. Both Facb and acid-treated IgG showed a fall in complement fixation relative to the native molecule of 30-40%.     

Interactions of divalent cations with phosphatidylserine bilayer membranes

The interaction of divalent cations with a homologous series of diacylphosphatidylserines (diacyl-PS) has been studied by differential scanning calorimetry and X-ray diffraction. Hydrated di-C14-PS (DMPS) exhibits a gel leads to liquid-crystal bilayer transition at 39 degrees C (delta H = 7.2 kcal/mol of DMPS). With increasing MgCl2 concentration, progressive conversion to a phase exhibiting a high melting (98 degrees C), high enthalpy (delta H congruent to 11.0 kcal/mol of DMPS) transition is observed. Similar behavior is observed for DMPS with increasing CaCl2 concentration. In this case, the high-temperature transition of the Ca2+-DMPS complex occurs at approximately 155 degrees C and is immediately followed by an exothermic transition probably associated with PS decomposition. For di-C12-, di-C14-, di-C16- (DPPS), and di-C18-PS, the transition temperatures of the Ca2+-PS complexes are in the range 151-155 degrees C; only di-C10-PS exhibits a significantly lower value, 142 degrees C. A different pattern of behavior is exhibited by DPPS in the presence of Sr2+ or Ba2+, with transitions in the range 70-80 degrees C being observed. X-ray diffraction of the Ca2+-PS complexes at 20 degrees C provides evidence of structural homology. All Ca2+-PS complexes exhibit bilayer structures, the bilayer periodicity increasing linearly from 35.0 A for di-C10-PS to 52.5 A for di-C18-PS. Wide-angle X-ray diffraction data indicate that hydrocarbon chain "crystallization" occurs on Ca2+-PS complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unfolding transitions of fibronectin and its domains. Stabilization and structural alteration of the N-terminal domain by heparin.

Changes in the conformational state of human plasma fibronectin and several of its fragments were studied by fluorescence emission, intrinsic fluorescence polarization and c.d. spectroscopy under conditions of guanidinium chloride-and temperature-induced unfolding. Fragments were chosen to represent all three types of internal structural homology in the protein. Low concentration (less than 2 M) of guanidinium chloride induced a gradual transition in the intact protein that was not characteristic of any of the isolated domains, suggesting the presence of interdomain interactions within the protein. Intermediate concentrations of guanidinium chloride (2-3 M) and moderately elevated temperatures (55-60 degrees C) induced a highly co-operative structural transition in intact fibronectin that was attributable to the central 110 kDa cell-binding domain. High temperatures (greater than 60 degrees C) produced a gradual unfolding in the intact protein attributable to the 29 kDa N-terminal heparin-binding and 40 kDa collagen-binding domains. Binding of heparin to intact fibronectin and to its N-terminal fragment stabilized the proteins against thermal unfolding. This was reflected in increased delta H for the unfolding transitions of the heparin-bound N-terminal fragment, as well as decreased accessibility to solvent perturbants of internal chromophores in this fragment when bound to heparin. These results help to account for the biological efficacy of the interaction between the fibronectin N-terminal domain and heparin, despite its relatively low affinity.     

Primary structure of the A chain of human complement-classical-pathway enzyme C1r. N-terminal sequences and alignment of autolytic fragments and CNBr-cleavage peptides.

Activated human complement-classical-pathway enzyme C1r has previously been shown to undergo autolytic cleavages occurring in the A chain [Arlaud, Villiers, Chesne & Colomb (1980) Biochim. Biophys. Acta 616, 116-129]. Chemical analysis of the autolytic products confirms that the A chain undergoes two major cleavages, generating three fragments, which have now been isolated and characterized. The N-terminal alpha fragment (approx. 210 residues long) has a blocked N-terminus, as does the whole A chain, whereas N-terminal sequences of fragments beta and gamma (approx. 66 and 176 residues long respectively) do not, and their N-terminal sequences were determined. Fragments alpha, beta and gamma, which are not interconnected by disulphide bridges, are located in this order within C1r A chain. Fragment gamma is disulphide-linked to the B chain of C1r, which is C-terminal in the single polypeptide chain of precursor C1r. CNBr cleavage of C1r A chain yields seven major peptides, CN1b, CN4a, CN2a, CN1a, CN3, CN4b and CN2b, which were positioned in that order, on the basis of N-terminal sequences of the methionine-containing peptides generated from tryptic cleavage of the succinylated (3-carboxypropionylated) C1r A chain. About 60% of the sequence of C1r A chain (440-460 residues long) was determined, including the complete sequence of the C-terminal 95 residues. This region shows homology with the corresponding parts of plasminogen and chymotrypsinogen and, more surprisingly, with the alpha 1 chain of human haptoglobin 1-1, a serine proteinase homologue.     

Improved efficiency of mutation detection by denaturing high-performance liquid chromatography using modified primers and hybridization procedure

The addition of a 20-base GC-clamp to a DNA fragment enabled mutations to be detected by denaturing high-performance liquid chromatography (DHPLC) in the higher melting domain of the two-domain fragment DYS271. The mutations were undetectable in the absence of the GC-clamp. The heteroduplex yield was greatly decreased by the presence of mutations in the high melting domain, presumably because this region anneals first during cooling, leading to selection of the more stable homoduplexes. Suppression of sequence-dependent melting behavior using betaine increased the heteroduplex yield almost four-fold. Mutations in the high melting domain were detected at 60 degrees C, whereas mutations in the low melting domain were detected at 56 degrees C. Computer modeling of the melting behavior agreed well with the experimental results, facilitating computer design of DHPLC amplicons.     

Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.

A denaturing gradient gel electrophoresis (DGGE) method is described that detects even single base pair changes in mitochondrial DNA (mtDNA). In this method, restriction fragments of mtDNA are electrophoresed in a urea/formamide gradient gel at 60 degrees C. Migration distance of each mtDNA fragment in the gel depends on melting behavior which reflects base composition. Fragments are located by Southern blotting with specific mtDNA probes. With just four carefully chosen restriction enzymes and as little as 50-100 ng of mtDNA, the method covers almost the entire human mitochondrial genome. To demonstrate the method, human mtDNA was analyzed. In six normal individuals, DGGE revealed melting behavior polymorphisms (MBPs) in mtDNA fragments that were not detected by restriction fragment length polymorphism (RFLP) analysis in agarose gels. Another individual, shown to have a melting behavior polymorphism in the cytochrome b coding region, was studied in detail. By mapping, the mutation was deduced to lie between nt 14905 and 15370. The affected fragment was amplified by PCR and sequenced. Specific base changes were identified in the region predicted by the gel result. This method will be especially useful as a diagnostic tool in mitochondrial disease for rapid localization of mtDNA mutations to specific regions of the genome, but DGGE also could complement RFLP analysis as a more sensitive method to follow maternal lineage in human and animal populations in a variety of research fields.