Ferenc Vonderviszt

Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling

The bacterial flagellar filament is a helical propeller constructed from 11 protofilaments of a single protein, flagellin. The filament switches between left- and right-handed supercoiled forms when bacteria switch their swimming mode between running and tumbling. Supercoiling is produced by two different packing interactions of flagellin called L and R. In switching from L to R, the intersubunit distance (∼52 Å) along the protofilament decreases by 0.8 Å. Changes in the number of L and R protofilaments govern supercoiling of the filament. Here we report the 2.0 Å resolution crystal structure of a Salmonella flagellin fragment of relative molecular mass 41,300. The crystal contains pairs of antiparallel straight protofilaments with the R-type repeat. By simulated extension of the protofilament model, we have identified possible switch regions responsible for the bi-stable mechanical switch that generates the 0.8 Å difference in repeat distance.

Terminal regions of flagellin are disordered in solution

Limited proteolysis of flagellin from Salmonella typhimurium SJW1103 by subtilisin, trypsin and thermolysin results in homologous degradation patterns. The terminal regions of flagellin are very sensitive to proteolysis. These parts are degraded into small oligopeptides at the very early stage of a mild digestion that yields a relatively stable fragment with a molecular weight of 40,000. Further proteolytic degradation results in a stable 27,000 Mr fragment. The 40,000 Mr tryptic fragment has been identified as residues 67 to 446 of the flagellin sequence, while the 27,000 Mr fragment involves the 179 to 418 segment. The NH2-terminal sequence positions for the corresponding fragments produced by subtilisin are 60 and 174 for the 40,000 Mr and 27,000 Mr fragments, respectively. The fragments lost their polymerizing ability. Structural properties of flagellin and its 40,000 Mr tryptic fragment were compared by circular dichroism spectroscopy and differential scanning calorimetry. Analysis of the calorimetric melting profiles suggests that terminal parts of flagellin have no significant internal stability and they are in extensive contact with water. However, these regions contain some secondary structure, probably alpha-helices, as revealed by comparison of the circular dichroic spectra in the far-ultraviolet region. Our results indicate that, although the terminal regions of flagellin may contain some alpha-helical secondary structure of marginal stability, they have no compact ordered tertiary structure in solution. On the contrary, the central region of the molecule involves at least two compact structural units.

Role of the disordered terminal regions of flagellin in filament formation and stability

Terminal regions of flagellin from Salmonella typhimurium, residues 1 to 65 and 451 to 494, have no ordered tertiary structure in solution, which makes them very susceptible to proteolytic degradation. Flagellin was subjected to mild controlled proteolytic treatment with highly specific proteases to remove terminal segments from the disordered regions. It is demonstrated here that various fragments can be readily prepared that differ from each other in 1 x 10(3) to 2 x 10(3) Mr segments in their NH2- or COOH-terminal regions. Terminally deleted fragments of flagellin were used to clarify the role of the disordered regions in the self-assembly of flagellin. The polymerization ability of the fragments was tested by inducing filament formation with ammonium sulfate. We found that fragments of flagellin containing large terminal deletions could form straight filaments, although the stability of these filaments required high salt concentrations. Even a fragment lacking the whole mobile COOH-terminal part of flagellin and 36 residues from the NH2-terminal region could form long filaments. The fragments could be also polymerized onto native flagellar seeds, suggesting that the subunit packing of the filaments of fragments is similar to that of the native ones. The fragments could also copolymerize with native flagellin, resulting in various helical forms. Filaments of fragments were found to be straight at both pH 4.0 and pH 12.5, indicating that they might have lost their polymorphic ability. Our results show that the major part of the disordered terminal regions of flagellin is not essential for polymerization, but it does play an important role in stabilization of the filaments and in influencing their polymorphic conformation.

Termini of Salmonella flagellin are disordered and become organized upon polymerization into flagellar filament

The terminal regions of Salmonella flagellin are essential for polymerization to form the flagellar filament. It has recently been suggested, on the basis of results from circular dichroism spectroscopy and scanning calorimetry, that these regions are disordered in solution. We report here direct evidence for disorder and mobility in the terminal regions of flagellin using 400 MHz proton nuclear magnetic resonance (n.m.r.) spectroscopy. Comparison of the n.m.r. spectra of monomeric and polymeric flagellin shows that the terminal regions become organized when polymerized to form the filament.

Structural organization of flagellin

The terminal regions of flagellin from Salmonella typhimurium have been reported to be disordered in solution, whereas the central part of the molecule contains protease-resistant, compact structural units. Here, conformational properties of flagellin and its proteolytic fragments were investigated and compared to characterize the domain organization and secondary structure of flagellin. Deconvolution analysis of the calorimetric melting profiles of flagellin and its fragments suggests that flagellin is composed of three co-operative units or domains. The central part of the molecule, residues 179 to 418, consists of two domains (G1 and G2), whereas the third domain (G3) is discontinuous, constructed from segments 67 to 178 and 419 to 448. Secondary structure prediction and analysis of far-ultraviolet circular dichroic spectra have revealed that G1 and G2 consist predominantly of beta-structure with a little alpha-helical content. G3 contains almost equal amounts of alpha and beta-structure, while in the terminal parts of flagellin the ordered secondary structure seems to be entirely alpha-helical.

Terminal disorder: A common structural feature of the axial proteins of bacterial flagellum?

We report, based on proteolytic experiments and high resolution 1H nuclear magnetic resonance studies that the terminal regions of the monomeric hook protein are highly mobile and exposed to the solvent. The disordered parts of the hook protein span approximately the first 70 and the last 30 amino acid residues. Although the amino acid sequences of flagellin and hook protein do not resemble each other at all, both proteins have now been shown to contain large disordered terminal regions. Sequential similarities of flagellin and hook protein, especially near the NH2 and COOH termini, to other axial components of bacterial flagellum suggest that terminal disorder may be a common structural feature of the axial proteins of the bacterial flagellum.

A possible way for prediction of domain boundaries in globular proteins from amino acid sequence

A simple approach to domain border prediction in globular proteins is outlined relying on the amino acid sequence only. Statistically determined sequential and association preference data of amino acids were combined to generate short range preference profiles along the polypeptide chains. Domain boundaries correlate with the minima of preference profiles, but some false minima also exist. Possibilities are discussed to exclude the false minima and to further improve the efficiency of the algorithm.

Change of geometry of polyacetylene upon charge transfer

Abstract Crystal orbital calculations on cis- and trans-polyacethylene chains indicate two kinds of geometry changes on charge transfer. (1) increase (decrease) of the CC bond lenght and CCC bond angles in the trans conformer taking up (losing) electrons, and (u) a tendency towards bond-length equalization on electron uptake and loss for both conformers.

Interaction of FliS flagellar chaperone with flagellin.

Premature polymerization of flagellin (FliC), the main component of flagellar filaments, is prevented by the FliS chaperone in the cytosol. Interaction of FliS with flagellin was characterized by isothermal titration calorimetry producing an association constant of 1.9 × 107 M−1 and a binding stoichiometry of 1:1. Experiments with truncated FliC fragments demonstrated that the C‐terminal disordered region of flagellin is essential for FliS binding. As revealed by thermal unfolding experiments, FliS does not function as an antifolding factor keeping flagellin in a secretion‐competent conformation. Instead, FliS binding facilitates the formation of α‐helical secondary structure in the chaperone binding region of flagellin.

Optical anisotropy of flagellin layers: In situ and label-free measurement of adsorbed protein orientation using OWLS

The surface adsorption of the protein flagellin was followed in situ using optical waveguide lightmode spectroscopy (OWLS). Flagellin did not show significant adsorption on a hydrophilic waveguide, but very rapidly formed a dense monolayer on a hydrophobic (silanized) surface. The homogeneous and isotropic optical layer model, which has hitherto been generally applied in OWLS data interpretation for adsorbed protein films, failed to characterize the flagellin layer, but it could be successfully modeled as an uniaxial thin film. This anisotropic modeling revealed a significant positive birefringence in the layer, suggesting oriented protein adsorption. The adsorbed flagellin orientation was further evidenced by monitoring the surface adsorption of truncated flagellin variants, in which the terminal protein regions or the central (D3) domain were removed. Without the terminal regions the protein adsorption was much slower and the resulting films were significantly less birefringent, implying that intact flagellin adsorbs on the hydrophobic surface via its terminal regions.