Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose.

The 9E10 antibody epitope (EQKLISEEDL) derives from a protein sequence in the human proto-oncogen p62(c-myc) and is widely used as a protein fusion tag. This myc-tag is a powerful tool in protein localization, immunochemistry, ELISA or protein purification. Here, we characterize the myc-tag epitope by substitutional analysis and length variation using peptide spot synthesis on cellulose. The key amino acids of this interaction are the core residues LISE. The shortest peptide with a strong binding signal is KLISEEDL. Dissociation constants of selected peptide variants to the antibody 9E10 were determined. scFv constructs with the shortest possible myc-tags were successfully detected by Western blot and ELISA, giving a signal comparable to that of the original myc-tag.

Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.

The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation. Implications to cyclization of peptides for stabilization are discussed.

Stability and DNA-binding properties of the omega regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035.

At the transcriptional level, the pSM19035-encoded omega protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type omega protein and a variant with a C-terminal hexa-histidine tag (omega-H(6)) were characterized. The omega protein is mainly alpha-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, T(m), between approximately 43 and approximately 78 degrees C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The omega-H(6) protein has a modified conformation with lower alpha-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.

Interaction of a designed interleukin-10 epitope mimic with an antibody studied by isothermal titration microcalorimetry.

The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.

The C-terminal subdomain (IF2 C-2) contains the entire fMet-tRNA binding site of initiation factor IF2.

Previous protein unfolding studies had suggested that IF2 C, the 24. 5-kDa fMet-tRNA binding domain of Bacillus stearothermophilus translation initiation factor IF2, may consist of two subdomains. In the present work, the four Phe residues of IF2 C (positions 531, 599, 657, and 721) were replaced with Trp, yielding four variant proteins having intrinsic fluorescence markers in different positions of the molecule. Comparison of the circular dichroism and Trp fluorescence changes induced by increasing concentrations of guanidine hydrochloride demonstrated that IF2 C indeed consists of two subdomains: the more stable N-terminal (IF2 C-1) subdomain containing Trp-599, and the less stable C-terminal (IF2 C-2) subdomain containing Trp-721. Isolated subdomain IF2 C-2, which consists of just 110 amino acids (from Glu-632 to Ala-741), was found to bind fMet-tRNA with the same specificity and affinity as native IF2 or IF2 C-domain. Trimming IF2 C-2 from both N and C termini demonstrated that the minimal fragment still capable of fMet-binding consists of 90 amino acids. IF2 C-2 was further characterized by circular dichroism; by urea-, guanidine hydrochloride-, and temperature-induced unfolding; and by differential scanning calorimetry. The results indicate that IF2 C-2 is a globular molecule containing predominantly beta structures (25% antiparallel and 8% parallel beta strands) and turns (19%) whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF2 C-1.

The fMet-tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues.

Mutations of the cysteines (positions 668 and 714) were generated in the IF2 C domain of Bacillus stearothermophilus translation initiation factor IF2. The corresponding proteins were characterized functionally and structurally. Most (yet not all) amino acid replacements at both positions resulted in severe reduction of the fMet-tRNA binding activity of IF2 C without grossly altering its structure. Our work demonstrates that: (a) both Cys residues are buried within an hydrophobic core and not accessible to protonation or chemical substitution, (b) neither Cys is functionally essential and (c) both Cys residues are located near the active site, probably without participating directly in fMet-tRNA binding.

Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments.

Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively).

Melting points of lysozyme and ribonuclease A crystals correlated with protein unfolding: a Raman spectroscopic study.

The effects of a temperature increase on monoclinic and tetragonal lysozyme single crystals were investigated by polarizing microscopy, X-ray diffraction and laser Raman spectroscopy. To prevent dissolution, the mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was observed around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and higher crystal forces in monoclinic lysozyme. Raman spectral changes in the amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature where birefringence changes, and differences in the S-S band suggest that in monoclinic lysozyme, denaturation is accompanied with disruption of some S-S bonds. Comparison with thermal denaturation and gel formation (beta-aggregation) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme crystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at the unfolding temperature at 333 K, i.e. loss of structure induces breakdown of crystal lattice and macroscopic shape. Although the crystal lattice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of thermal unfolding of the polypeptide chains.

C repeats of the streptococcal M1 protein achieve the human serum albumin binding ability by flanking regions which stabilize the coiled-coil conformation.

The M and M-like proteins of Streptococcus pyogenes are fibrous cell surface proteins. They have multiple binding sites for several human proteins and are composed of the C-terminal anchor domain, the alpha-helical coiled-coil domain, and the N-terminal non-coiled-coil domain. The coiled-coil domain of the M1 protein consists of repeat units called B, C, and D and a spacer unit S between B and C. Recombinant fragments A-B-S-C-D, A-B-S, B-S-C, S-C, S-C-D, C-D, and C of the coiled-coil domain were studied by analyzing their secondary structures and binding affinities to human serum albumin (HSA). As shown by circular dichroism, all fragments are in an alpha-helical conformation. C-D and S-C-D form coiled coils at room temperature and bind below 37 degrees C with high affinity to HSA. C-D and S-C-D unfold in two steps with Tm values of approximately 31 and approximately 65 degrees C; complex formation with HSA increases the unfolding temperatures. B-S-C has a lower alpha-helical content, a less pronounced coiled-coil conformation, and a reduced thermal stability, binds HSA weaker, and is only slightly stabilized by HSA binding in comparison to C-D and S-C-D. C and S-C are less stable than the other fragments and are not organized as coiled coils showing some features of alpha-helical single strands only below 20 degrees C, and binding of HSA was not observed. The results indicate that the formation of coiled-coil structures, supported by flanking D regions and, to a lesser extent also B regions, is essential for the binding of C repeat units to HSA.

Conformation and stability of streptokinases from nephritogenic and nonnephritogenic strains of streptococci.

Conformation and stability of three Sks from Streptococcus equisimilis strain H46A, Streptococcus pyogenes strain A374, and Streptococcus pyogenes strain AT27 were compared by limited proteolysis, CD, and fluorescence measurements and by DSC. The general similarity of the peptide CD spectra in the spectral region 185 to 260 nm indicates the same type of folding for the three proteins. Fluorescence and aromatic CD spectra are consistent with a predominant surface localization of the aromatic amino acids and a low rigidity of their surroundings. A major difference among the three Sks is shown by deconvolution of their excessive heat capacity functions. Deconvolution reveals two energetic folding units in Sk H46A but three energetic folding units in Sk A374 and Sk AT27. Digestion of the Sks with trypsin indicates a reduced sensitivity of the C-terminal region of Sk A374 and Sk AT27 in comparison to Sk H46A. This suggests that amino acids of the C-terminal region participate in the formation of the third folding unit of Sk A374 and Sk AT27.

Individual amino acids in the N-terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases.

Thermostability and unfolding behavior of the wild-type (1,3-1,4)-beta-glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12-M) delta F14 and H(A12-M) delta Y13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12-M) delta F14 is constructed by the fusion of 12 N-terminal amino acids of AMY with amino acids 13-214 of MAC, and by deletion of F14. In H(A12-M) delta Y13F14A, the N-terminal region of MAC is exchanged against the AMY sequence, Y13 is deleted, and Phe 14 is exchanged against Ala. The sequence of the N-terminal loop region from Pro 9 to amino acid 16 (or 17) is very important for the properties of the enzymes and influences the effects of Ca2+ ions on the thermostability and unfolding behavior of the enzymes. The half transition temperatures T(m) are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca(2+)-free buffer, MAC, H(A12-M) delta F14 and H(A12-M) delta Y13F14A unfold in a single cooperative transition from the folded state to the unfolded state, whereas for AMY, a two-step unfolding was found. In the presence of Ca2+, the two-step unfolding of AMY is strengthened. Furthermore, for H(A12-M) delta F14, a two-step unfolding is induced by Ca2+. These data indicate a two-domain structure of AMY and H(A12-M) delta F14, in the presence of Ca2+. Thus, point mutations in a peripheral loop region are decisive for thermal stabilities and unfolding mechanisms of the studied glucanases in the presence of Ca2+.

Chemical modification of recombinant HIV-1 capsid protein p24 leads to the release of a hidden epitope prior to changes of the overall folding of the protein.

It was found that the affinity of a monoclonal antibody directed against a recombinantly expressed HIV-1 capsid protein p24 (rp24) strongly increased after chemical modification of the Iysine residues of rp24 with different amounts of maleic anhydride. The extent and the sites of modification were analyzed by MALDI-TOF mass spectrometry. Unmodified rp24 and the differently modified rp24 samples were tested for binding the murine monoclonal antibody CB4-1 which recognizes the epitope GATPQDLNTML comprising residues 46-56 of rp24. An increase in the number of modified lysine residues led to enhanced binding affinity of CB4-1. Most pronounced effects were observed after substitution of the first amino groups: an average number of three modified residues per protein molecule increases the binding affinity by a factor of 23, but the substitution of the remaining nine residues increases the binding affinity only by a factor of 11. Fully modified rp24 variant proteins were bound by CB4-1 with Kd values comparable to that of the peptide epitope. Conformation and stability of the unmodified rp24, highly (rp24F, 9 residues; rp24G, 11 residues) modified, and fully modified protein (rp24I, 11 lysine residues and N-terminus) were analyzed by circular dichroism (CD) and fluorescence spectroscopy under different solvent conditions. Little difference in conformation and unfolding behavior was observed between the unmodified and highly modified rp24, which differ drastically in the antibody binding behavior. The fully modified sample, however, displayed a significant decrease in alpha-helical content. Thus, the epitope seems to be hidden (cryptotope) in the unmodified rp24 in a low-affinity binding conformation and becomes displayed at low levels of chemical modification which obviously induce subtle structural changes prior to changes of the overall folding observable by spectroscopic means.

Conformation and stability of recombinant HIV-1 capsid protein p24 (rp24).

Conformation and stability of the recombinant protein HIV-1 rp24 were analyzed by circular dichroism, fluorescence spectroscopy and differential scanning calorimetry under different solvent conditions. From circular dichroism measurements, HIV-1 rp24 at pH 5.8 can be classified as an all alpha-helical protein. A fluorescence maximum of about 330 nm indicates a predominantly hydrophobic environment of the five tryptophan residues. The GdnHCl-induced unfolding curves monitored by CD and fluorescence are sigmoidal and single phasic and the midpoints of transitions are independent on the protein concentration. For the calculation of free energy of unfolding delta GuH2O a 'two-state' model was applied. The calculated values are between 18 and 24 kJ/mol and thus on the lower limit of the conformational stability of globular proteins. Melting experiments at pH 5.8 are impaired by a strong irreversible aggregation at higher temperatures. However, at pH 3.0 and in the presence of 0.1% (w/v) ocytl beta-glucopyranoside the melting curves show a large degree of reversibility with a Tm value of 38 degrees C and a molar enthalpy change delta Hm of 218 kJ/mol. At pH < 2.5 HIV-1 rp24 can adopt a new conformation which is characterized by a high alpha-helical content, a strongly decreased CD in the aromatic region, a red-shift of the fluorescence spectrum and a strong binding of ANS. These spectral features of the acid-induced conformational state are similar to those obtained for molten globule-like folding states. HIV-1 rp24 unfolds cooperatively at pH 2.0 in the concentration range of about 1.5-3.0 M GdnHCl. The calculated values delta GuH2O at pH 2.0 of about 12 kJ/mol are significantly decreased in comparison to the delta GuH2O values of the protein at pH 5.8.

Influence of Ca2+ on conformation and stability of three bacterial hybrid glucanases.

The three hybrid glucanases (1-12)AMY x MAC(13-214), (1-12)AMY x des-Tyr13MAC(14-214); (1-16)AMY x MAC(17-214) are composed of short N-terminal segments of 12 or 16 amino acid residues derived from the Bacillus amyloliquefaciens glucanase (AMY) and of residues 13-214, 14-214 and 17-214, respectively, derived from the Bacillus macerans enzyme (MAC). The three proteins have similar conformational features as shown by the similar characteristics of their CD spectra in the far- and near-ultraviolet region. A metal-ion-binding site was identified in the hybrid glucanase (1-16)AMY x MAC(17-214) by a crystal structure analysis [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291]. Only minor conformational changes of the three hybrid glucanases were observed depending on the presence or absence of Ca2+ ions but for (1-16)AMY x MAC(17-214) and (1-12)AMY x des-Tyr13MAC(14-214) the occupation of this metal-binding site by a Ca2+ ion is connected with a large increase of the stability against thermal and chemical unfolding. Surprisingly, for (1-12)AMY x MAC(13-214), which differs from (1-12)AMY x des-Tyr13MAC(14-214) by only one additional amino acid in an N-terminal loop region, the effect of Ca2+ ions on the stability is small. The exchange of a few amino acid residues near the N-terminus of the B. macerans glucanase against amino acids found at comparable positions in the B. amyloliquefaciens glucanase seems to influence very strongly the strength of the Ca2+ binding site and concomitantly the stability of the hybrid glucanases.

Stability of Escherichia coli single-stranded DNA binding protein (EcoSSB)

Conformation and stability of EcoSSB, a single-stranded DNA binding protein encoded by Escherichia coli, were analyzed by circular dichroism and fluorescence measurements. From CD measurements at pH 7.5, EcoSSB can be classified as a protein with high alpha-helix and beta-sheet content. The hydrophobicity of the environment of the tryptophan residues of the native protein is only marginally increased in comparison to the unfolded protein. The GdnHCl induced unfolding curves measured by CD and fluorescence are coincident and sigmoidal and show a monophasic transition. The stability of EcoSSB is concentration dependent and the unfolding behavior can be described as a two-state transition from the folded tetrameter to unfolded monomers. The mean values of free energy of dissociation and unfolding delta GH2O mu are between 173 and 177 kJ.mol-1 and the mean half concentration c1/2 of GdnHCl of the transition curves are about 1.5 M and 1.7 M for protein concentrations of 0.1 mg.ml-1 and 0.5 mg.ml-1, respectively.

Conformations and stabilities of human Glu1- and Lys78-plasminogen and of the fragments mini- and microplasminogen, analysed by circular dichroism and differential scanning calorimetry.

The conformations and stabilities of two forms of human plasminogen, Glu1-plasminogen (Glu1-HPg, Glu1-Asn791) and Lys78-plasminogen (Lys78-HPg, Lys78-Asn791), and two enzymatically derived plasminogen fragments, miniplasminogen (mini-HPg, Val443-Asn791) and microplasminogen (micro-HPg, Lys531-Asn791) were analysed by circular dichroism and differential scanning calorimetry. The two plasminogen forms differ by the lack of 77 N-terminal amino acids in Lys78-HPg in comparison to Glu1-HPg. Mini-HPg is composed of kringle 5 and the protease domain of HPg whereas micro-HPg is built from the protease domain of HPg and a stretch of about 15 amino acids from kringle 5. Differential scanning calorimetric measurements of Glu1-HPg and Lys78-HPg reveal seven thermal transitions for both plasminogen forms. The results obtained for Lys78-HPg largely agree with recently published data (Novokhatny, V. V., Kudinov, S. A. and Privalov, P. L. J. Mol. Biol. 1984, 179, 215). Three thermal transitions corresponding to kringle 5 and to two subdomains of the C-terminal protease region were identified for mini-HPg. In micro-HPg, the two thermal transitions of the protease region were found but one of the protease subdomains was modified and its stability was much higher than in any of the other studied proteins. According to the microcalorimetric data obtained for mini-HPg and micro-HPg, transitions 5 and 6 of Glu1-HPg and Lys78-HPg were reassigned to kringle 5 and to a subdomain of the protease region, respectively, in contrast to literature data.(ABSTRACT TRUNCATED AT 250 WORDS)

Microcalorimetric determination of the thermostability of three hybrid (1-3,1-4)-beta-glucanases.

Thermodynamic parameters of the three hybrid (1-3,1-4)-beta-glucanases H(A12-M), H(A12-M) delta Y13, and H(A16-M) composed of short N-terminal regions derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the homologous Bacillus macerans enzyme were determined in 2 mM sodium cacodylate pH 6.0, 1.5M guanidine hydrochloride, containing 1 mM CaCl2 or 1 mM EDTA. Melting of H(A12-M) delta Y13 and H(A16-M) in the presence of calcium ions is characterized by two subtransitions; only one transition is observed in the case of H(A12-M). In calcium-free buffer each of the three hybrid enzymes melts in one two-state transition. Transition temperatures Tm and molar enthalpy changes delta H are reduced in the absence of calcium ions but the reduction is much more pronounced for H(A12-M) delta Y13 and H(A16-M) than for the less thermostable enzyme H(A12-M).

Conformations and conformational changes of four Phe-->Trp variants of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis studied by circular dichroism and fluorescence spectroscopy.

Circular dichroic spectra in the region 180-260 nm of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis and of four mutants with a Phe residue replaced by Trp, i.e. [F29W]HBsu, [F47W]HBsu, [F50W]HBsu and [F79W]HBsu, show minor differences only and demonstrate the general similarity of the conformations of these proteins. Fluorescence maxima at 315-320 nm and 330-335 nm indicate a more hydrophobic environment or a more effective stacking of Trp residues in mutants [F29W]HBsu and [F50W]HBsu in comparison to [F47W]HBsu and [F79W]HBsu, respectively. Unfolding of the mutants in high-ionic-strength buffers by increasing concentrations of urea results in a red shift of the fluorescence emission maxima to about 350 nm; the fluorescence intensities decrease strongly for [F29W]HBsu and [F50W]HBsu but show a small increase for [F47W]HBsu and [F79W]HBsu. The data suggest complex unfolding patterns with subtle differences between the single mutants. The circular dichroic spectra in the region 250-320 nm are dominated by the effects of the Trp residues and signal position-dependent differences in the environment of the Trp residues. The conformations of the mutant proteins depend on the ionic strength of the buffer and become more stable against unfolding by denaturants or increasing temperatures at higher ionic strength. At low ionic strength a pronounced protein-concentration dependence of the conformation of the mutants is seen.

Conformational stability of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis, and of the four HBsu variants [F29W], [F47W], [F50W] and [F79W].

From denaturation studies with urea a free energy delta GuH2O of unfolding of 49.8 kJ.mol-1 at 25C was calculated for the histone-like DNA-binding protein HBsu from Bacillus subtilis. Unfolding was monitored by circular dichroism measurements observing the changes of the molar mean residue ellipticity [theta] at 222 nm. For the calculation of delta Gu a two-state model of unfolding, i.e. the unfolding of native dimers into unfolded monomers, was applied. The validity of this model in high ionic strength buffer was proven by measurements at different protein concentrations yielding the same delta Gu values. Four HBsu variants, each carrying one single point mutation ([F29W], [F47W], [F50W] and [F79W]) were analysed with respect to their stability against unfolding at increasing temperatures and urea concentrations. The delta Gu values of mutants were calculated using the two-state model and show a reduced stability of the variants [F29W], [F47W], [F50W] and [F79W] in comparison to the wild type HBsu with delta delta Gu values of -9.2 kJ.mol-1, -7.5 kJ.mol-1, -5.9 kJ.mol-1, and -7.5 kJ.mol-1, respectively. Similar delta delta Gu values were obtained for the HBsu mutant proteins by thermal unfolding experiments.

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.