Non-native Helices Research Articles

Optimized periphery-core interface increases fitness of the Bacillus subtilis glmS ribozyme.

Like other functional RNAs, ribozymes encode a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-periphery interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (k-seq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. The k-seq results and folding assays showed that most deleterious mutations lower activity by impairing ribozyme self-assembly. All-atom molecular dynamics simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop introduce a non-native tertiary interface that rewires the catalytic center, eliminating activity. We conclude that the need to avoid non-native helix packing powerfully constrains the evolution of tertiary structure motifs in RNA.

Non-native Helix lucorum Linnaeus, 1758 (Gastropoda: Eupulmonata: Helicidae) after twelve years in Prague, Czech Republic

The first occurrence of Helix lucorum Linnaeus in the Czech Republic was reported 12 years ago, at the closed train station Žižkov in Prague. A part of the station is a ruderal habitat while large patches are covered with partly damaged concrete. At the site where it was first recorded, and where the density of H. lucorum is still the highest, this invasive snail has now almost completely replaced the original H. pomatia Linnaeus. However, it has not expanded either inside or outside the station area.

Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix

In the folding of β-lactoglobulin (βLG), a predominantly β-sheet protein, a transient intermediate possessing an excess amount of non-native α-helix is formed within a few milliseconds. To characterize the early folding dynamics of βLG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native α-helix formation within 200μs. Time-resolved SAXS showed that the radius of gyration (Rg) of the intermediate at 300μs was 23.3±0.7Å, indicating a considerable collapse from the unfolded state having Rg of 35.1±7.1Å. Further compaction to Rg of 21.2±0.3Å occurred with a time constant of 28±11ms. Pair distribution functions showed that the intermediate at 300μs comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of α-helix but similar Rg of 23.0±0.8Å, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native α-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of βLG.

Effect of non-native helix destabilization on the folding of equine β-lactoglobulin.

β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.

Transient non-native helix formation during the folding of β-lactoglobulin.

In ideal proteins, only native interactions are stabilized step-by-step in a smooth funnel-like energy landscape. In real proteins, however, the transient formation of non-native structures is frequently observed. In this review, the transient formation of non-native structures is described using the non-native helix formation during the folding of β-lactoglobulin as a prominent example. Although β-lactoglobulin is a predominantly β-sheet protein, it has been shown to form non-native helices during the early stage of folding. The location of non-native helices, their stabilization mechanism, and their role in the folding reaction are discussed.

Contribution to the knowledge of terrestrial molluscs in southeastern Ukraine

Records of terrestrial molluscs collected in nine scattered sites in southeastern Ukraine are presented and discussed. The findings of non-native Helix lucorum and Lucilla singleyana are especially interesting. Coloration of H. lucorum in different parts of its range seems to be significantly different. A role of keel in species determination of collected Helicopsis sp. shells is discussed. Variability of Deroceras subagreste coloration is described. New data on a species composition of terrestrial molluscs in several protected areas is given.

Misplaced helix slows down ultrafast pressure-jump protein folding

Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 μs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 μs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.

A Native Disulfide Stabilizes Non-Native Helical Structures in Partially Folded States of Equine β-Lactoglobulin

Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non-native α-helices are present in two different regions, residues 98-107 and 114-135, and are connected by a native disulfide bond. The CD spectra of two peptides that correspond to the helical regions are characterized by weak helical signatures, and the sum of their CD spectra is nearly the same as the spectrum of disulfide-reduced CHIBLΔF. Therefore, the non-native helices are stabilized by the disulfide, and non-native helix formation may occur only during the refolding of the disulfide-intact protein. Supporting this conclusion is the observation that tear lipocalin, a homologue of ELG that lacks the disulfide, does not form non-native helices during folding.

Non‐native α‐helix formation is not necessary for folding of lipocalin: Comparison of burst‐phase folding between tear lipocalin and β‐lactoglobulin

Tear lipocalin and beta-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of beta-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence.

The Stabilization Mechanism of the Intermediate Structure of Equine Beta-Lactoglobulin

A single-disulfide mutant of equine β-lactoglobulin, C66A/C160A, forms an expanded and helical conformation at an acidic pH and a low anion concentration (C state). The C state is a model of an early folding intermediate of βlactoglobulin. The secondary structures in the C state are in the regions corresponding to F, G, H strands and the major α-helix, and they assume the native-like or nonnative helices. Peptides encompassing those helices did not form stable helices. A longer fragment, CHIBL, which encompasses the structured region in the C states, has a helical structure similar to the corresponding region of the full-length protein in the C state. This result indicates that non-local interactions responsible for the helix formation in the C state reside in the sequence of CHIBL. The disulfide bond, Cys106-Cys119, links two nonnative helices in the C state. This is one of the possible long-range interactions stabilizing the helical structures. The CD spectrum of disulfide-reduced C66A/C160A shows decreased helical content. This indicates that the helices are stabilized by this disulfide bond. The detailed structural analysis of the reduced C66A/C160A by using proline mutants suggests that the nonnative helix in the G strand region can form without the disulfide bond despite Cys106 is contained in that region. It also suggests that the disulfide bond is crucial to the formation of the nonnative helix in the H strand region. We synthesized a peptide encompassing two helices formed in the C state. The GssH peptide is disulfide-linked G-peptide and H-peptide. This peptide did not form stable helical structures. Therefore G and H strands and major helix regions must interact with each other simultaneously to form stable helices in the C state.

Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli

One of the necessary conditions for a protein to be foldable is the presence of a complete set of "folding elements" (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE-assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE-FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non-native helices. MD simulations also show the non-native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE-FE interactions, are also one of the necessary conditions for a protein to be foldable.

Glutamine 53 is a gatekeeper residue in the FK506 binding protein.

The effect of non-random conformational averaging in the urea-unfolded state on the folding pathway has been investigated in a variant of the FK506 binding protein with three additional residues at the amino terminus (FKBP(*)). Three mutations (asparagine, aspartate, and threonine) were introduced into position Q53 to enhance formation of non-native helix observed in this part of the protein in the urea-unfolded state. NMR analysis showed minor structural changes in the native state of each mutant, but additional medium-range alphaN(i,i+2) of each mutant nuclear Overhauser enhancements were observed in the urea-unfolded state that were not in FKBP(*), indicating that the mutations had a more substantial effect on the unfolded state ensemble than on the native state ensemble. Isothermal equilibrium denaturation measurements showed that the Q53T and Q53D mutants were destabilized, whereas the Q53N mutant was stabilized relative to FKBP(*) with little change in the equilibrium m values. The unfolding rates of Q53N and Q53T were similar to that of FKBP(*), but Q53D unfolded twice as fast as FKBP(*). In contrast, the mutations had a more pronounced effect on the refolding kinetics. Q53N refolded slightly faster and exhibited a kinetic folding intermediate similar to that of FKBP(*). The Q53D and Q53T mutants also refolded faster than FKBP(*) but lacked the folding intermediate, indicating that these mutants experienced a different folding trajectory and transition state than FKBP(*) and Q53N. The refolding kinetic Phi values were 0.74, 1.4 and 7.9 for Q53N, Q53T, and Q53D, respectively. The data point to Q53 functioning as a gatekeeper residue in the folding of FKBP(*). This study shows that perturbing the unfolded state ensemble via mutagenesis can provide insights into residues that play important roles in the folding pathway, and represents an attractive strategy for mapping the high-energy portions of the folding energy landscape.

α → β transition of β-lactoglobulin as evidenced by heteronuclear NMR

Whereas bovine β-lactoglobulin is a predominantly β-sheet protein, it has a marked α-helical preference and can be considered to be a useful model of the α → β transition, a key issue for understanding the folding and biological function of a number of proteins. In order to understand the mechanism of the α → β transition, the backbone structures of the recombinant bovine β-lactoglobulin A in the native state and in the highly helical state induced by 2,2,2-trifluoroethanol were characterized by 1H, 13C and 15N multidimensional NMR spectroscopy. Overall, the secondary structures in the native state were similar to those of the crystal structure. On the other hand, β-lactoglobulin in the 2,2,2-trifluoroethanol state was composed of many α-helical segments. The presence of the persistent α-helices in the helical state and the core β-sheet in the native state suggested that during folding native-like core β-sheet and several non-native helices are formed first and the remaining β-sheet is subsequently “induced” through interaction with the pre-existing β-sheet.