A synthetic mimic of a discontinuous binding site on interleukin-10.

We synthetically reconstructed a discontinuous binding site on interleukin-10 (IL-10) that recognizes the neutralizing anti-IL-10 antibody CB/RS/1. To design the 32-mer IL-10 mimic, a discontinuous interaction site on IL-10 was mapped, and binding studies with epitope-derived peptides led to specific replacement of several amino acids. Both parts of the interaction site were combined by addition of a linker molecule. Systematic analoging of the combined molecule then led to introduction of several additional substitutions in both regions and the linker. All possible disulfide bridge-containing variants of the 32-mer were tested by binding studies. Parallel syntheses were performed on continuous cellulose membranes by spot synthesis. As a result, a conformationally stabilized IL-10-derived molecule was obtained that both binds to and neutralizes the biological activity of CB/RS/1 in the low nanomolar range. This synthetic approach is a powerful alternative to phage display methods for the design of protein mimics.

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions.

1. By enlarging the dimensions of the gels used in the usual analytical two-dimensional polyacrylamide gel electrophoresis it is possible to separate much larger amounts of ribosomal protein in comparison to analytical separations. 15 mg of protein mixture of small or large subunits of rat liver ribosomes can be separated by this procedure. 2. The positions of the proteins in the two-dimensional patterns are identified with a special staining procedure. The proteins are eluted from the gels with SDS/phosphate buffers. 3. The purity of the extracted proteins was tested by one-dimensional SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis, respectively. 24 proteins of the small and 24 proteins of the large ribosomal subunit were isolated in pure form. 4. The amino acid compositions of 24 proteins of the small and of 19 proteins of the large subunit were determined.

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.

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).

A small-angle and wide-angle x-ray scattering study of the shape and secondary structure of native 5 S RNA from rat liver ribosomes.

Native 5 S RNA from rat liver ribosomes was investigated by means of small-angle and wide-angle X-ray scattering. The radius of gyration, Rs of the molecule is 3.1 nm, the maximum dimension, L, 10.5 nm and the shape volume, Vc, about 60 nm3. The overall shape of the molecule as derived from these parameters is a flat elliptical cylinder with dimensions of 2a = 10.45 nm, 2b = 6.94 nm, H = 1.06 nm. The cross-section radius of gyration of 0.92 nm and the mass per unit length of 2610 mol X nm-1 of the 5 S RNA molecule are typical for a double helical organized RNA molecule. From wide-angle scattering data it can be concluded that the double helical regions are in the A-form characterized by an 11-fold helix with a turn angle of 32.7 degrees and a distance of 0.28 nm between adjacent base pairs. A refined electron density model of a distorted L shape is proposed for the 5 S RNA molecule.

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.

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+.

Laser Raman studies of the 5 S rRNA-protein L5 complex of rat liver ribosomes.

The effects of ribosomal protein L5 on the conformation of 5 S rRNA in the 5 S rRNA-protein L5 complex extracted from rat liver ribosomes have been studied by laser Raman spectroscopy. A comparison of the spectra shows small protein-induced conformational changes in the 5 S rRNA, but most of the base-paired regions appear to be present in the complex with protein L5 as well as in the free 5 S rRNA. Furthermore specific interactions between 5 S rRNA and protein L5 are indicated. Cytosine (and/or uracil) residues in single-stranded regions and the N(7) of guanine are engaged in interactions with the protein as suggested by the Raman data.

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.

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 fMet-tRNA(fMet) with the C-terminal domain of translational initiation factor IF2 from Bacillus stearothermophilus.

Analytical ultracentrifugation studies indicated that the C-terminal domains of IF2 comprising amino acid residues 520-741 (IF2 C) and 632-741 (IF2 C-2) bind fMet-tRNA with similar affinities (K(d) at 25 degrees C equal to 0.27 and 0.23 microM, respectively). Complex formation between fMet-tRNA(fMet) and IF2 C or IF2 C-2 is accompanied by barely detectable spectral changes as demonstrated by a comparison of the Raman spectra of the complexes with the calculated sum of the spectra of the individual components. These results and the temperature dependence of the K(d) of the protein-RNA complexes indicate that complex formation is not accompanied by obvious conformational changes of the components, and possibly depends on a rather small binding site comprising only a few interacting residues of both components.

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.

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.

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).

X-ray structure of the cyclomaltohexaicosaose triiodide inclusion complex provides a model for amylose-iodine at atomic resolution.

Cyclomaltohexaicosaose (CA26) is folded into two 1(2)/(3) turns long V-helices that are oriented antiparallel. Crystals of complexes of CA26 with NH(4)I(3) and Ba(I(3))(2) are brown and X-ray analyses show that I(3)(-) units are located in the approximately 5 A wide central channels of the V-helices. In the complex with NH(4)I(3), two CA26 molecules are stacked to form 2 x 1(2)/(3) turns long channels harbouring 3 I(3)(-) at 3.66-3.85 A inter I(3)(-) distance (shorter than van der Waals distance, 4.3 A), whereas in the Ba(I(3))(2) complex, CA26 are not stacked and only one I(3)(-) each fills the V-helices. Glucose...I contacts are formed with C5-H, C3-H, C6-H and (at the ends of the V-helices) with O6 in (+) gauche orientation. By contrast, O2, O3, O4 and O6 in the preferred (-) gauche orientation do not interact with I because these distances are >/=4.01 A and exceed the van der Waals I...O sum of radii by about 0.5 A except for one O2...I distance of 3.68 A near the end of one V-helix. Raman spectra indicate that the complexes share the presence of I(3)(-) with blue amylose-iodine.

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)

Conformational properties of streptokinase--differential scanning calorimetric investigations.

The two-domain structure of streptokinase (Sk) was demonstrated by scanning calorimetric investigations at neutral pH and low ionic strength. The melting pattern of the protein is composed of two two-state transitions at TtrS1 = 45.9 +/- 0.4 degrees C with delta H1 = 431 +/- 18 kJ/mol, and TtrS2 = 60.1 +/- 1.3 degrees C with delta H2 = 306 +/- 16 kJ/mol. The partial specific heat capacity of native Sk was determined to be Cp = 1.42 +/- 0.17 J/K/g and the denaturational heat capacity change associated with the two transitions, delta Cp1 = 0.21 J/K/g and delta Cp2 = 0.38 J/K/g, respectively. The overall melting pattern of Sk remains almost unchanged at a variety of tested solvent compositions, except at pH 4 (and below) and in the presence of denaturants. The two domains show different susceptibility to urea. It is proposed that the less thermostable domain is located within the N-terminal part (residues 1-230), and the more thermostable one, within the C-terminal region.

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.

Conformational properties of streptokinase--secondary structure and localization of aromatic amino acids.

The conformational properties of streptokinase (Sk) have been assessed by several spectroscopic techniques. A solvent accessibility of about 70% of the 22 Tyr residues was found by u.v. perturbation spectroscopy. Fluorescence spectroscopy indicates also the surface localization of the single Trp 6 residue. Circular dichroism (c.d.), infrared (i.r.), and Raman spectra were analysed in order to estimate the contents of secondary structure elements of Sk. Values in the range of 14-23% alpha-helices, 38-46% beta-structures, 10-30% turns and 12-23% residual structures were found. The characteristics of the c.d. spectrum support the classification of Sk as an alpha + beta protein. Effects of temperature, pH, and denaturants were studied by c.d. spectroscopy, and on spin-labelled Sk, by e.p.r. spectroscopy. Structural effects were induced at temperatures above 40 degrees C, pH values below 3.0 and urea concentrations above 2 M. At temperatures above 70 degrees C, at pH 2.1, and at urea and Gu.HCl concentrations of 7 M and 5 M, respectively, no further structural changes are revealed in the spectra. At temperatures around 50 degrees C, at pH 3.0, and denaturant concentrations of about 1 M Gu.HCl and 1 M to 2 M urea, c.d. effects were observed in the near-u.v. region indicating an increase in the asymmetry for aromatic amino acids in comparison with the structure of Sk in low ionic strength buffers at neutral pH, 20 degrees C and in the absence of denaturants. These effects were most pronounced for the temperature dependence of the c.d. spectra. E.p.r. spectroscopy has shown that loosening of the protein surrounding of the spin label already begins at 1 M urea and that the mobility of the spin label points to a structural change in Sk at 46 degrees C.