Heinz Welfle

Quasielastic light scattering from human α-lactalbumin: comparison of molecular dimensions in native and ‘molten globule’ states

Abstract Quasielastic light scattering has been applied to compare the linear dimensions of human α-lactalbumin molecules in the native ‘molten globule’ and unfolded states. The translational diffusion coefficients of the protein have been measured at neutral and acid pH as well as in 6 m guanidine hydrochloride. Temperature dependence of diffusion coefficients for Ca2+-free protein at neutral and acid pH have also been obtained. After correction for the protein association, it is shown that the effective linear dimensions of protein molecules increase by about 10% in the ‘molten globule’ state (i.e. at acid pH or high temperature) as compared with the native state. This increase in linear dimensions is much smaller than the increase in the unfolded state (∼40%

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.

In vitro and in vivo stability of the epsilon2zeta2 protein complex of the broad host-range Streptococcus pyogenes pSM19035 addiction system

Abstract Streptococcus pyogenes pSM19035-encoded (10.7 kDa) and ζ (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with ζ acting as toxin that kills plasmiddeprived cells and as an antitoxin that neutralises the activity of ζ. The and ζ proteins copurify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as 2ζ2 heterotetramer in solution. Cocrystals of the 2ζ2 complex contain and ζ in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the ζ protein has a significantly lower thermodynamic stability than the protein both in free state and in the complex. Proteolytic studies indicate that ζ protein is more stable in the the 2ζ2 complex than in the free state. In vivo studies reveal a short halflife of the antitoxin (~18min) and a long lifetime of the ζ toxin (>60min). When transcriptiontranslation of a plasmid containing the and ζ genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the protein from the background.

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 β structures (25% antiparallel and 8% parallel β strands) and turns (19%) whose structural properties are not grossly affected by the presence or absence of the N-terminal subdomain IF2 C-1.

Secondary structure of globulins from plant seeds: a re-evaluation from circular dichroism measurements

Abstract The secondary structure parameters of plant seed globulins (11S from Brassica napus L, 11S from Helianthus annuus L, IIS from Vicia faba , 7S from Phaseolus vulgaris L) have been determined from their circular dichroism spectra by the method of Provencher and Glockner. According to this method, the proteins contain 40–50% β-sheet structure and only about 10% helical structure. We conclude, therefore, that the plant seed globulins belong to the class of β-sheet proteins. Their overall secondary structure is homologous. It is shown that the method of Provencher and Glockner provides reasonable secondary structure parameters for proteins which are rich in β-sheet structure even if the spectral range utilized for analysis is restricted to 210–240 nm.

Interaction of fMet-tRNAfMet 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°C equal to 0.27 and 0.23 μM, respectively). Complex formation between fMet‐tRNAfMet 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.

STRUCTURAL ANALYSIS OF THE A AND B CONFORMERS OF ESCHERICHIA COLI 5 S RIBOSOMAL RNA BY INFRARED SPECTROSCOPY

1. Introduction Rapid progress is being made in elucidating the structure of pro- and eukaryotic 5 S RNAs using dif- ferent physical, biochemical and sequence analysis approaches (reviewed in [ 11). It seems to be evident from comparative sequence studies [l-5] that a general base pairing scheme of the type first proposed in [2] is valid for eukaryotic 5 S RNAs and that a general secondary structure of the type first proposed in [3] extended by few base pairs is the structural basis for prokaryotic 5 S RNAs. Experimental evi- dence supporting these basic secondary structures is now manifold and derives, e.g., from such powerful specific techniques as high-resolution ‘H NMR spec- troscopy [6,7] and slow tritium exchange studies [8]. Summarizing data from optical ([6,9] and references within), infrared [lo], Raman [l l-131, and ‘H NMR spectroscopy [6,7] it became obvious that both for prokaryotic and eukaryotic 5 S RNA molecules in solution a highly ordered secondary/tertiary structure exists with an amount of about 35-42 base pairs (58-70% of all nucleotides are base-paired) in the presence of stabilizing ions. and the infrared thermal melting curves reveal partic- ular differences between both conformers at 570°C which may be useful with respect to the analysis of the intricate problem of the A-to-B conformational transition(s). 2. Materials and methods Here, we report the results of infrared spectro- scopic studies of the A and B conformers of

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.

Role of the N-terminal region and of beta-sheet residue Thr29 on the activity of the omega2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035.

Abstract The dimeric regulatory protein wild-type ω (wt ω2) binds to arrays of 7-bp sequences (heptads) present in the operator DNA region of copy control and partition functions of plasmid pSM19035. Each ω2 protein probably binds with an antiparallel β-sheet structure in the major groove of the 7-bp subsite of the operator DNA. Exchange of threonine at position 29 to alanine (T29A) drastically affects the activity of variant protein ω2T29A both in vivo and in vitro, and reduces the thermodynamic stability ΔGu0, but does not change the conformation. Likewise, the binding affinity to DNA is reduced and the association of the two monomeric subunits of the ω2T29A dimer is weakened, as manifested by an increase in the dissociation constant from 3.2 μM for wt ω2 to 6.3 μM for ω2T29A. Denatured dimers are formed upon thermal unfolding of wt ω2 and ω2T29A at ca. 45 μM (Dn↔Du). Removal of 8 (ω2ΔN8), or even 18 (ω2ΔN18) N-terminal amino acids has no obvious effect either on the core structure or on the activity in comparison to wt ω2. The stability of variants ω2ΔN8 and ω2ΔN18 is similar to that of wt ω2, and their binding to operator DNA is not impaired.

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering

The structures of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500±4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume Vc, surface Oc, radius of gyration Rs, maximum dimension of the molecule L, thickness D, and cross section radius of gyration Rsq, agree within the experimental error limits. The mean values are Vc=57±3 nm3, Oc=165±10 nm2, Rs=3.37±0.05 nm, L=10.8±0.7 nm, D=1.57±0.07 nm, Rsa=0.92±0.01 nm.Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution function. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D20,w0=(5.9±0.2)·10-7 cm2s-1 and (6.2±0.2)·10-7 cm2s-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively, a solvation shell of about 0.3 nm thickness around both molecules is determined. This structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest that all 5S rRNAs may indeed have conserved essentially the same type of folding of their polynucleotide strands during evolution, despite having very different sequences.