Mechanics of bacterial macrofiber initiation.

The twisting and writhing during growth of single-cell filaments of Bacillus subtilis which lead to macrofiber formation was studied in both left- and right-handed forms of strains FJ7 and RHX. Filament bending, touching, and loop formation (folding), followed by winding up into a double-strand fiber, were documented. Subsequent folds that produced multistrandedness were also examined. The rate of loop rotation during winding up was measured for 26 loops from 16 clones. In most cases, the first loop formed turned at a lower rate than those produced by the following cycles of folding. The sequence of folding topologies differed in FJ7 and RHX strains and in left- versus right-handed structures. Right-handed FJ7 routinely gave rise to four-stranded helices at the second fold, whereas left-handed FJ7 and both left-handed and right-handed forms of RHX made structures with predominantly two double-stranded helical regions. Left-handed RHX structures frequently produced second folds within the initial loop itself, resulting in T- or Y-shaped fibers. Sixteen cases in which the initial touch of a filament to itself produced a loop that snapped open before it could wind up into a double-strand fiber were found. The snap motions were used to obtain estimates of the forces generated by helical growth of single filaments and to investigate theoretical models involving the material properties of cell filaments. In general, the mechanical behavior of growing single-cell filaments and fibers consisting of two-, three-, or four-strand helices was similar to that described for larger, mature, multifilament macrofibers. The behavior of multicellular macrofibers can be understood, therefore, in terms of individual cell growth.     

Macrofiber structure and the dynamics of sickle cell hemoglobin crystallization

Fibers of deoxyhemoglobin S undergo spontaneous crystallization by a mechanism involving a variety of intermediate structures. These intermediate structures, in common with the fiber and crystal, consist of Wishner-Love double strands of hemoglobin S molecules arranged in different configurations. The structure of one of the key intermediates linking the fiber and crystal, called a macrofiber, has been studied by a variety of analytical procedures. The results of the analysis indicate that the intermediates involved in the fiber to crystal transition have many common structural features. Fourier analysis of electron micrographs of macrofibers confirms that they are composed of Wishner-Love double strands of hemoglobin molecules. Electron micrographs of macrofiber cross-sections reveal that the arrangement of the double strands in macrofibers resembles that seen in micrographs of the a axis projection of the crystal. This orientation provides an end-on view of the double strands which appear as paired dumb-bell-like masses. The structural detail becomes progressively less distinct towards the edge of the particle due to twisting of the double strands about the particle axis. Serial sections of macrofibers confirm that these particles do indeed rotate about their axes. The twist of the particle is right handed and its average pitch is 10,000 Å. The effect of rotation on the appearance of macrofiber cross-sections 300 to 400 Å thick can be simulated by a 15 ° rotation of an a axis crystal projection. The relative polarity of the double strands in macrofibers and crystals can be determined easily by direct inspection of the micrographs. In both macrofibers and crystals they are in an anti-parallel array.On the basis of these observations we conclude that crystallization of macrofibers involves untwisting and alignment of the double strands.     

Clockwise and counterclockwise pinwheel colony morphologies of Bacillus subtilis are correlated with the helix hand of the strain

Helical macrofiber-producing strains of Bacillus subtilis grown on fresh complex medium semisolid surfaces formed "pinwheel"-shaped colonies. Clockwise pinwheel projections arose from colonies of strains that produce right-handed helical macrofibers in fluid cultures. Most strains able to make left-handed helical macrofibers in fluid grew as disorganized wavy colonies without directed projections. A phage-resistant left-handed mutant was found that produces very tight colonies with pinwheel projections that lie counterclockwise relative to the colony. The pinwheel colony morphology is interpreted therefore in terms of the cell surface organization and helical growth.     

Characterization of nutrition-induced helix hand inversion of Bacillus subtilis macrofibers.

The kinetics of Bacillus subtilis macrofiber helix hand inversion was examined. Inversion was induced by transfer of structures produced in one medium to another medium. When cultured at 20 degrees C in either medium, the doubling time was approximately 100 min. To establish a baseline, the macrofiber twist state produced in one medium was measured over the same time course during which other macrofibers underwent inversion after transfer to a second medium. The baseline was used to identify the time of inversion initiation: the point at which curves representing changes of twist as a function of time after transfer to the new medium intersected the baseline. Right- and left-handed macrofibers of different twists were produced by growth in mixtures of TB and S1 media. These were used to determine the influence of initial twist on the time course of inversion initiation. In the right to left inversion, a positive correlation was found between initial twist and the time of inversion initiation. The left to right inversion differed, however, in that a constant time was required for inversion initiation regardless of the starting left-handed twist. When a nutritional pulse was administered by transferring fibers from TB to S1 to TB medium, the time to initiation of inversion was found to decrease with incubation of increasing duration in S1 medium. A similar pulse protocol was used in conjunction with inhibitors to examine the protein and peptidoglycan synthesis requirements for the establishment of nutrition-induced memory that leads to initiation of inversion. Nutritionally induced right to left inversion but not left to right inversion required protein synthesis. The addition of trypsin to left-handed macrofibers apparently required, as described previously for the temperature-regulated twist system (D. Favre, D. Karamata, and N. H. Mendelson, J. Bacteriol. 164:1141-1145, 1985), for the production of left-handed twist states in the nutrition system.     

Helical macrofiber formation in Bacillus subtilis: inhibition by penicillin G.

The folding process required for helical macrofiber formation after the outgrowth of Bacillus subtilis spores was found to be blocked by very low concentrations of penicillin G (1 to 3 ng/ml). Under such conditions, growth and septation without cell separation resulted in characteristic disorganized multicellular structures. Higher concentrations (4 and 10 ng/ml) were needed to inhibit spore outgrowth and vegetative growth, respectively.     

Structural analysis of polymers of sickle cell hemoglobin. II. Sickle hemoglobin macrofibers

Sickle cell hemoglobin macrofibers are an important intermediate in the low pH crystallization pathway of deoxygenated hemoglobin S that link the fiber to the crystal. Macrofibers are a class of helical particles differing primarily in their diameters but are related by a common packing of their constituent subunits. We have performed three-dimensional reconstructions of three types of macrofibers. These reconstructions show that macrofibers are composed of rows of Wishner-Love double strands in an arrangement similar to that in the crystal. We have measured the orientation and co-ordinates of double strands in macrofibers using cross-correlation techniques. In this approach, the electron density projections of double strands calculated from the known high-resolution crystal structure are compared with regions along the length of the particles in which the distinct pattern of double strands in c-axis projection may be observed. Contrary to assertions by Makinen & Sigountos (1984), our results unambigously demonstrate that adjacent rows of double strands in macrofibers are oriented in an antiparallel manner, as in the Wishner-Love crystal. Adjacent rows of antiparallel double strands are displaced along the helical axis relative to their co-ordinates in the crystal. Electron density models of macrofibers based on the crystallographic structure of the sickle hemoglobin double strand are in good agreement with the projections of macrofibers observed in electron micrographs. We have studied the structure of a closely related crystallization intermediate, the sickle hemoglobin paracrystal. The arrangement of double strands in paracrystals is similar to that in Wishner-Love crystals, except that they are displaced along the a-axis of the crystal. Measurements of the double strand co-ordinates reveal that the distribution of strand positions is bimodal. These results further establish the close structural relationship between macrofibers and paracrystals as intermediates in the crystallization of deoxygenated sickle hemoglobin.     

Identification of Mutations Associated with Macrofiber Formation in BACILLUS SUBTILIS

A search was made for the genes responsible for the production of helical macrofibers in the original collection of macrofiber-producing strains of B. subtilis. Two loci were identified: fibA, located between hisA and tag-1, and fibB, linked to cysB. fibA governs a short-lived division suppression phenomenon associated with the production of rudimentary fibers, whereas fibB appears to be responsible for a persistent division suppression and a more highly organized helical macrofiber. Both mutations are recovered from each of the original macrofiber-producing strains which also carried the div IV-B1 mutation responsible for minicell production. The latter mutation by itself is not sufficient, however, for the production of macrofibers. Other known mutations leading to division suppression that map in the same region are shown not to be allelic to fibA or fibB. Neither fib locus appears to be responsible for helix hand determination.     

Extended secondary structure in 5S rRNAs from a sulphur metabolizing archaebacterium, Thermococcus celer

While this sequence shares a significant homology with the 5S RNAs of other archaebacteria and is consistent with current models for the secondary structure of 5S RNAs, it contains three unusual features. The G + C content (72-74%) is significantly higher than other 5S RNAs; the secondary structure is distinguished by unusually stable and extended helical structures and, most important, there is evidence for sequence heterogeneity in the form of complementary base substitutions and precursor processing. This supports recent evidence (Newmann, H., Gierl, A., Tu, J., Leibrock, J., Staiger, D. and Zillig, W. (1983) Mol. Gen. Genet. 192, 66-72) that, like many of the higher eukaryotes, this group of sulphur-metabolizing bacteria may contain multiple 5S RNA genes.     

Regulation of Bacillus subtilis macrofiber twist development by ions: effects of magnesium and ammonium.

The steady-state twist of Bacillus subtilis macrofibers produced by growth in complex medium was found to vary as a function of the magnesium and ammonium concentrations. Four categories of macrofiber-producing strains that differed in their response to temperature regulation of twist were studied. Macrofibers were cultured in the complex medium TB used in previous experiments and in two derivative media, T (consisting of Bacto Tryptose), in which most strains produced left-handed structures, and Be (consisting of Bacto Beef Extract), in which right-handed macrofibers arose. In nearly all cases, increasing concentrations of magnesium led to the production of macrofibers with greater right-handed twist. Some strains unable to form right-handed structures as a function of temperature could be made to do so by the addition of magnesium. Inversion from right- to left-handedness in strain FJ7 induced by temperature shift-up was blocked by the addition of magnesium. The presence of magnesium during a high-temperature pulse did not block the establishment of "memory," although it delayed the initiation of the transient inversion following return to low temperature. The twist state of macrofibers grown without a magnesium supplement was not instantaneously affected by the addition of magnesium. Such fibers were, however, protected from lysozyme attack and associated relaxation motions. Lysozyme degradation of purified cell walls (both intact and lacking teichoic acid) was also blocked by the addition of magnesium. Ammonium ions influenced macrofiber twist development towards the left-hand end of the twist spectrum. Macrofiber twist produced in mixtures of magnesium and ammonium was strain and medium dependent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Bacillus subtilis macrofiber twist development by D-alanine.

Twist states of Bacillus subtilis macrofibers were found to vary as a function of the concentration of D-alanine in the medium during growth. L-Alanine in the same concentration range had no effect. Increasing concentrations of D-alanine resulted in structures progressively more right-handed (or less left-handed). All strains examined in this study, including mutants fixed in the left-hand domain as a function of temperature, responded to D-alanine in the same way. All twist states from tight left- to tight right-handedness could be achieved solely by varying the D-alanine concentration. The D-alanine-requiring macrofiber strain 2C8, which carries a genetic defect (dal-1) in the alanine racemase, behaved in a similar fashion. The combined effects of D-alanine and ammonium sulfate (a factor known to influence macrofiber twist development in the leftward direction) were examined by using both strains able to undergo temperature-induced helix hand inversion and others incapable of doing so. In all cases, the effects of D-alanine predominated. A synergism was found in which increasing the concentration of ammonium sulfate in the presence of D-alanine enhanced the right-factor activity of the latter. A D-alanine pulse protocol provided evidence that structures undergo a transient inversion indicative of "memory." Chloramphenicol treatment inhibited the establishment of memory in the D-alanine-induced right to left inversion, supporting the existence of a "left twist protein(s)" that is required for the attainment of left-handed twist states. Chemical analysis of cell walls obtained from right- and left-handed macrofibers produced in the presence and absence of D-alanine, respectively, failed to reveal twist state-specific differences in the overall composition of either peptidoglycan or wall teichoic acids.     

Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus

Background

Bacterial macrofibers twist as they grow, writhe, supercoil and wind up into plectonemic structures (helical forms the individual filaments of which cannot be taken apart without unwinding) that eventually carry loops at both of their ends. Terminal loops rotate about the axis of a fiber's shaft in contrary directions at increasing rate as the shaft elongates. Theory suggests that rotation rates should vary linearly along the length of a fiber ranging from maxima at the loop ends to zero at an intermediate point. Blocking rotation at one end of a fiber should lead to a single gradient: zero at the blocked end to maximum at the free end. We tested this conclusion by measuring directly the rotation at various distances along fiber length from the blocked end. The movement of supercoils over a solid surface was also measured in tethered macrofibers.

Results

Macrofibers that hung down from a floating wire inserted through a terminal loop grew vertically and produced small plectonemic structures by supercoiling along their length. Using these as markers for shaft rotation we observed a uniform gradient of initial rotation rates with slopes of 25.6°/min. mm. and 36.2°/min. mm. in two different fibers. Measurements of the distal tip rotation in a third fiber as a function of length showed increases proportional to increases in length with constant of proportionality 79.2 rad/mm. Another fiber tethered to the floor grew horizontally with a length-doubling time of 74 min, made contact periodically with the floor and supercoiled repeatedly. The supercoils moved over the floor toward the tether at approximately 0.06 mm/min, 4 times faster than the fiber growth rate. Over a period of 800 minutes the fiber grew to 23 mm in length and was entirely retracted back to the tether by a process involving 29 supercoils.

Conclusions

The rate at which growing bacterial macrofibers rotated about the axis of the fiber shaft measured at various locations along fibers in structures prevented from rotating at one end reveal that the rate varied linearly from zero at the blocked end to maximum at the distal end. The increasing number of twisting cells in growing fibers caused the distal end to continuously rotate faster. When the free end was intermittently prevented from rotating a torque developed which was relieved by supercoiling. On a solid surface the supercoils moved toward the end permanently blocked from rotating as a result of supercoil rolling over the surface and the formation of new supercoils that reduced fiber length between the initial supercoil and the wire tether. All of the motions are ramifications of cell growth with twist and the highly ordered multicellular state of macrofibers.     

Temperature-pulse-induced "memory" in Bacillus subtilis macrofibers and a role for protein(s) in the left-handed-twist state

Macrofibers in steady-state growth at one temperature were subjected to pulses of various durations at a temperature at which the opposite helix hand would form and then returned to the initial temperature. In an upshift pulse (20 to 48 degrees C), at least 3 min of incubation was required to induce a transient inversion that occurred later after return to 20 degrees C. Longer pulses resulted in shorter delays in onset of the transient inversion. This "memory" of a brief high-temperature pulse suggests that even a small amount of material can influence the twist of the entire macrofiber. Similar results were found for temperature downshift pulses corresponding to the opposite inversion. Adding chloramphenicol during the temperature pulse blocked the establishment of memory associated with the right-to-left inversion but not that associated with left-to-right inversion. In contrast, inhibiting peptidoglycan synthesis with D-cycloserine during the temperature pulse did not prevent establishment of memory. Inhibiting protein synthesis in mutants fixed as left-handed structures over the entire temperature range induced conversion to right-handedness but did not affect mutants fixed as right-handed structures. Adding protease to either live or formaldehyde-killed macrofibers always induced rotations of right-handed orientation. Steady-state growth in the presence of protease was found to shift the initial macrofiber twist towards the right-hand end of the twist spectrum. The phenomenon was observed in several mutants with different initial twists.     

Helical junctions as determinants for RNA folding: origin of tertiary structure stability of the hairpin ribozyme

Helical junctions are ubiquitous structural elements that govern the folding and tertiary structure of RNAs. The tobacco ringspot virus hairpin ribozyme consists of two helix-loop-helix elements that lie on adjacent arms of a four-way junction. In the active form of the hairpin ribozyme, the loops are in proximity. The nature of the helical junction determines the stability of the hairpin ribozyme tertiary structure [Walter, N. G., Burke, J. M., and Millar, D. P. (1999) Nat. Struct. Biol. 6, 544-549] and thus its catalytic activity. We used two-, three-, and four-way junction hairpin ribozymes as model systems to investigate the thermodynamic basis for the different tertiary structure stabilities. The equilibrium between docked and extended conformers was analyzed as a function of temperature using time-resolved fluorescence resonance energy transfer (trFRET). As the secondary and tertiary structure transitions overlap, information from UV melting curves and trFRET had to be combined to gain insight into the thermodynamics of both structural transitions. It turned out that the higher tertiary structure stability observed in the context of a four-way junction is the result of a lower entropic cost for the docking process. In the two- and three-way junction ribozymes, a high entropic cost counteracts the favorable enthalpic term, rendering the docked conformer only marginally stable. Thus, two- and three-way junction tertiary structures are more sensitive toward regulation by ligands, whereas four-way junctions provide a stable scaffold. Altogether, RNA folding and stability appear to be governed by principles similar to those for the folding of proteins.     

Inversion of helix orientation in Bacillus subtilis macrofibers

The ability of helical macrofibers of Bacillus subtilis to convert from left- to right-handed structures or vice versa has been known to be controlled by the nutritional environment (N. H. Mendelson, Proc. Natl. Acad. Sci. U.S.A., 75:2478-2482, 1978). lyt mutants (Ni15, FJ3, FJ6, and FJ7) and also lyt phenocopies of wild-type strain FJ8 were able to undergo helix hand inversion as a function of temperature. The transition between right- and left-handed structures was in a very narrow range (about 2.5 degrees C) in the low to mid-40 degrees C. The helix orientation of these strains was also influenced by the concentration of divalent ions. Macrofiber handedness is governed, therefore, by at least four factors: genetic composition, temperature, and nutritional and ionic environments. Conditions normally used for growth fall, within this matrix, in the region favoring right-handed structures. Inhibition studies suggest that cell growth must occur for helix hand inversion.     

The origin of the rotation of one end of a cell relative to the other end during growth of gram-positive rods

The Gram-positive rod wall elongates by an inside-to-outside mechanism of linking new peptidoglycan on the inside and the cracking, by autolysis, of old wall on the outside. During this process the peptidoglycan experiences stress in different directions in different levels of the wall. The stress that develops in a rod-shaped cell if the wall was uniform in physical properties throughout its thickness is twice as great in the hoop direction as in the axial direction. This leads to splitting in the direction of the longitudinal axis. However, the older, partially split, more peripheral wall is stressed in the direction of the elongating cell axis and thus favors circumferential cracks. It is suggested that these processes combine to form a system of helical cracks, grooves, or crevasses. The stable system of grooves would have the same handedness, fairly constant pitch and elongate as the cell grows. Their continuing development would result in the rotation of one end of the cell relative to the other even in cells with no spiral or apparent helical character. Such rotation has been experimentally observed with Bacillus subtilis. The proposed mechanism for rotation during growth may account, in part, for the formation of helical coils of bundles of filamentous organisms (macrofibers), the morphology of spirilla and vibroids, and for the shapes of some mutant and some antibiotic-treated organisms. Rotation due to generation of helical cracks as the result of the biophysics of the growth process as proposed here, is an alternative to the proposal by Mendelson (1976, Helical growth of Bacillus subtilis: a new model for cell growth. Proc. natn. Acad. Sci. U.S.A. 73, 1740-1744) that rotation is due to the laying down of nascent peptidoglycan in a helical pattern.     

Folding of apocytochrome c in lipid micelles: formation of alpha-helix precedes membrane insertion.

Apocytochrome c, which in aqueous solution is largely unstructured, acquires a highly alpha-helical structure upon interaction with lipid. The alpha-helix content induced in apocytochrome c depends on the lipid system, and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The folding kinetic mechanism of apocytochrome c induced by zwitterionic micelles of lysophosphatidylcholine (L-PC), predominantly driven by hydrophobic lipid-protein interactions, was investigated by fluorescence stopped-flow measurements of Trp 59 and fluorescein-phosphatidylethanolamine-(FPE) labeled micelles, in combination with stopped-flow far-UV circular dichroism. It was found that formation of the alpha-helical structure of apocytochrome c precedes membrane insertion. The unfolded state in solution (U(W)) binds to the micelle surface in a helical conformation (I(S)) and is followed by insertion into the lipid micelle, i.e., formation of the final helical state H(L). Binding of apocytochrome c to the lipid micelle (U(W) --> I(S)) is concurrent with formation of a large fraction (75-100%, depending on lipid concentration) of the alpha-helical structure of the final lipid-inserted state H(L). The highly helical intermediate I(S) is formed on the time scale of 3-12 ms, depending on lipid concentration, and inserts into the lipid micelle (I(S) --> H(L)) in the time range of approximately 200 ms to >1 s, depending on lipid-to-protein ratio. The final lipid-inserted helical state H(L) in L-PC micelles has an alpha-helix content approximately 65% of that of cytochrome c in solution and has no compact stable tertiary structure as revealed by circular dichroism results.     

Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.     

Assignment of proton resonances, identification of secondary structural elements, and analysis of backbone chemical shifts for the C102T variant of yeast iso-1-cytochrome c and horse cytochrome c

Resonance assignments for the main-chain, side-chain, exchangeable side chain, and heme protons of the C102T variant of Saccharomyces cerevisiae iso-1-cytochrome c in both oxidation states (with the exception of Gly-83) are reported. (We have also independently assigned horse cytochrome c.) Some additional assignments for the horse protein extend those of Wand and co-workers [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry 28, 186-194; Feng, Y., Roder, H., Englander, S. W., Wand, A. J., & Di Stefano, D. L. (1989) Biochemistry 28, 195-203]. Qualitative interpretation of nuclear Overhauser enhancement data allows the secondary structure of these two proteins to be described relative to crystal structures. Comparison of the chemical shift of the backbone protons of the C102T variant and horse protein reveals significant differences resulting from amino acid substitution at positions 56 and 57 and further substitutions between residue 60 and residue 69. Although the overall folding of yeast iso-1-cytochrome c and horse cytochrome c is very similar, there can be large differences in chemical shift for structurally equivalent residues. Chemical shift differences of amide protons (and to a lesser extent alpha protons) represent minute changes in hydrogen bonding. Therefore, great care must be taken in the use of differences in chemical shift as evidence for structural changes even between highly homologous proteins.     

Solution conformation of a peptide fragment representing a proposed RNA-binding site of a viral coat protein studied by two-dimensional NMR

The first 25 amino acids of the coat protein of cowpea chlorotic mottle virus are essential for binding the encapsidated RNA. Although an alpha-helical conformation has been predicted for this highly positively charged N-terminal region [Argos, P. (1981) Virology 110, 55-62; Vriend, G., Verduin, B. J. M., & Hemminga, M. A. (1986) J. Mol. Biol. 191, 453-460], no experimental evidence for this conformation has been presented so far. In this study, two-dimensional proton NMR experiments were performed on a chemically synthesized pentacosapeptide containing the first 25 amino acids of this coat protein [Ten Kortenaar, P. B. W., Krüse, J., Hemminga, M. A., & Tesser, G. I. (1986) Int. J. Pept. Protein Res. 27, 401-413]. All resonances could be assigned by a combined use of two-dimensional correlated spectroscopy and nuclear Overhauser enhancement spectroscopy carried out at four different temperatures. Various NMR parameters indicate the presence of a conformational ensemble consisting of helical structures rapidly converting into more extended states. Differences in chemical shifts and nuclear Overhauser effects indicate that lowering the temperature induces a shift of the dynamic equilibrium toward more helical structures. At 10 degrees C, a perceptible fraction of the conformational ensemble consists of structures with an alpha-helical conformation between residues 9 and 17, likely starting with a turnlike structure around Thr9 and Arg10. Both the conformation and the position of this helical region agree well with the secondary structure predictions mentioned above.     

The helix clock: a potential biomechanical cell cycle timer

A model based upon helical geometry that provides cylindrically shaped cells with a means to measure their length during growth and to time cell cycle events is presented. The helix clock arises from the change in pitch angle that accompanies the parallel packing of strands on a cylinder surface. A single strand inserted into the cylinder surface nearly perpendicular to the long axis of the cylinder starts the clock running. As additional strands are inserted parallel to those in place, the pitch angle of all strands must reorient. A limit is reached when all strands lie parallel to the long axis of the cylinder. By sensing either the pitch angle or a physical ramification thereof, cells can measure their length during growth and time events of the cell cycle. The helix clock model is discussed in relationship to the bacterial cell cycle. The idea that bacterial cells use one helix hand for cylinder elongation, the other for septation is presented. The negative twist so generated apparently drives folding in the helical bacterial macrofiber system of Bacillus subtilis.