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1.
Abstract Adaptor properties of linear hairpin helices have been examined. The analysis suggests that neither right nor left handed hairpin helices can simultaneously read a comma free messenger and align aminoacyl residues for peptide condensation. Comparison of these studies with the model of the present day peptidyl transfer intermediate suggests that the “L” shaped folding of the present day tRNAs may be a prerequisite for adaptor function. Therefore, the three-dimensional organization of the ancestral adaptor molecule must have had structural features similar to its present day counterpart. 相似文献
2.
The pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven alpha-helix bundle (alpha1-alpha7). According to the "umbrella model," alpha4 and alpha5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, alpha4-loop-alpha5, its alpha4 and alpha5 helices, as well as mutant alpha4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the alpha4-loop-alpha5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of alpha4-loop-alpha5 as the membrane-inserted pore-forming hairpin in which alpha4 and alpha5 line the lumen of the channel and alpha5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active alpha4 mutant peptide completely inhibits alpha4-loop-alpha5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an "immunity protein" by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well. 相似文献
3.
R. Rein S. Srinivasan J. McDonald G. Raghunathan M. Shibata 《Origins of life and evolution of the biosphere》1987,17(3-4):431-438
The molecular mechanisms underlying the primitive translational apparatus have been studied in light of present day protein
biosynthesis. Using the structural information available from the contemporary system as a key to its function, both the structural
necessities for an early adaptor and the multipoint recognition properties of such adaptors have been investigated. This was
done by first critically examining the potential feasibility of right- and left-handed hairpin adaptor models. Second, a molecular
model of the contemporary transpeptidation complex has been constructed in order to ascertain the structural requirements
of the adaptor molecule needed for peptidyl transfer. Third, a model of the tRNATyr-tyrosyl tRNA synthetase complex including the positioning of the disordered region is proposed. This model is used to illustrate
those required recognition properties of aminoacyl synthetase which lead to a perspective on the structure of the ancestor
synthetase. 相似文献
4.
Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed. 相似文献
5.
6.
We have recently reported a first experimental turn propensity scale for transmembrane helices. This scale was derived from measurements of how efficiently a given residue placed in the middle of a 40 residue poly(Leu) stretch induces the formation of a "helical hairpin" with two rather than one transmembrane segment. We have now extended these studies, and have determined the minimum length of a poly(Leu) stretch compatible with the formation of a helical hairpin. We have also derived a more fine-grained turn propensity scale by (i) introducing each of the 20 amino acid residues into the middle of the shortest poly(Leu) stretch compatible with helical hairpin formation, and (ii) introducing pairs of residues in the middle of the 40 residue poly(Leu) stretch. The new turn propensities are consistent with the amino acid frequencies found in short hairpin loops in membrane proteins of known 3D structure. 相似文献
7.
The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns. 相似文献
8.
Helix-helix interactions within membranes are dominated by van der Waals packing motifs and side chain-side chain hydrogen bond formation, which act in tandem to determine the residues that comprise the interface between two given helices. To explore in a systematic manner the tertiary contacts between transmembrane helices, we have designed and expressed in Escherichia coli highly hydrophobic helix-loop-helix constructs of prototypic sequence K(1)KKKKKKFAIAIAIIAWAX(19)AIIAIAIAIKSPGSKIAIAIAIIAZ(44)AWAIIAIAIAFKKKKKKK(62), where "small" (Ala) and "large" (Ile) residues were used to maximize the tertiary contact area. Evidence that the two transmembrane (TM) segments in the AI construct contain an interface conducive for folding into a hairpin structure was obtained from the results that (i) the single TM AI(pep) peptide derived from the AI hairpin forms SDS-resistant dimers on PAGE gels and (ii) the corresponding sequence forms a strong dimer when examined in vivo in TOXCAT assays. Site-directed mutagenesis of AI hairpins was carried out to incorporate each of the 20 commonly occurring amino acids at X positions. Analysis on Western blots using an oligomerization assay in 12% NuPage-sodium dodecyl sulfate (SDS) indicated that mutants with X = E, D, Q, R, N, H, and K largely formed SDS-resistant dimers-which likely correspond to H-bonded four-helix bundles-while all the others (e.g., X = F, W, L, I, M, V, C, Y, A, T, S, G, and P) remained monomeric. Systematic studies of X/Z double mutants indicated that formation of hairpin dimers is the result of the disruption of stabilizing interactions between the antiparallel helices within the AI construct. The overall results suggest that, in situations where hydrophobic van der Waals packing energy between helices is sufficient to prevent significant rotation about the major axes of interacting helices, intrahairpin side chain-side chain H-bond formation will occur mainly when pairs of polar residues are interfacially located and proximal. Knowledge of the relative contributions of these forces should be of value, for example, in clarifying the context--and the structural consequences--of disease-related mutations. 相似文献
9.
Unusual mRNA pseudoknot structure is recognized by a protein translational repressor 总被引:27,自引:0,他引:27
Translation of ribosomal proteins in the alpha operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression. 相似文献
10.
Colicins are water-soluble toxins that, upon interaction with membranes, undergo a conformational change, insert, and form pores in them. Pore formation activity is localized in a bundle of 10 α-helices named the pore-forming domain (PFD). There is evidence that colicins attach to the membrane via a hydrophobic hairpin embedded in the core of the PFD. Two main models have been suggested for the membrane-bound state: penknife and umbrella, differing in regard to the orientation of the hydrophobic hairpin with respect to the membrane. The arrangement of the amphipathic helices has been described as either a compact three-dimensional structure or a two-dimensional array of loosely interacting helices on the membrane surface. Using molecular dynamics simulations with an implicit membrane model, we studied the structure and stability of the conformations proposed earlier for four colicins. We find that colicins are initially driven towards the membrane by electrostatic interactions between basic residues and the negatively charged membrane surface. They do not have a unique binding orientation, but in the predominant orientations the central hydrophobic hairpin is parallel to the membrane. In the inserted state, the estimated free energy tends to be lower for the compact arrangements of the amphipathic helix, but the more expanded ones are in better agreement with experimental distance distributions. The difference in energy between penknife and umbrella conformations is small enough for equilibrium to exist between them. Elongation of the hydrophobic hairpin helices and membrane thinning were found unable to produce stabilization of the transmembrane configuration of the hydrophobic hairpin. 相似文献
11.
Rudresh Ramakumar S Ramagopal UA Inai Y Goel S Sahal D Chauhan VS 《Structure (London, England : 1993)》2004,12(3):389-396
De novo design of supersecondary structures is expected to provide useful molecular frameworks for the incorporation of functional sites as in proteins. A 21 residue long, dehydrophenylalanine-containing peptide has been de novo designed and its crystal structure determined. The apolar peptide folds into a helical hairpin supersecondary structure with two right-handed helices, connected by a tetraglycine linker. The helices of the hairpin interact with each other through a combination of C-H.O and N-H.O hydrogen bonds. The folding of the apolar peptide has been realized without the help of either metal ions or disulphide bonds. A remarkable feature of the peptide is the unanticipated occurrence of an anion binding motif in the linker region, strikingly similar in conformation and function to the "nest" motif seen in several proteins. The observation supports the view for the possible emergence of rudimentary functions over short sequence stretches in the early peptides under prebiotic conditions. 相似文献
12.
Dynamic Monte Carlo simulations of globular protein folding/unfolding pathways. II. Alpha-helical motifs 总被引:1,自引:0,他引:1
Dynamic Monte Carlo simulations of the folding pathways of alpha-helical protein motifs have been undertaken in the context of a diamond lattice model of globular proteins. The first question addressed in the nature of the assembly process of an alpha-helical hairpin. While the hairpin could, in principle, be formed via the diffusion-collision-adhesion of isolated performed helices, this is not the dominant mechanism of assembly found in the simulations. Rather, the helices that form native hairpins are constructed on-site, with folding initiating at or near the turn in almost all cases. Next, the folding/unfolding pathways of four-helix bundles having tight bends and one and two long loops in the native state are explored. Once again, an on-site construction mechanism of folding obtains, with a hairpin forming first, followed by the formation of a three-helix bundle, and finally the fourth helix of the native bundle assembles. Unfolding is essentially the reverse of folding. A simplified analytic theory is developed that reproduces the equilibrium folding transitions obtained from the simulations remarkably well and, for the dominant folding pathway, correctly identifies the intermediates seen in the simulations. The analytic theory provides the free energy along the reaction co-ordinate and identifies the transition state for all three motifs as being quite close to the native state, with three of the four helices assembled, and approximately one turn of the fourth helix in place. The transition state is separated from the native conformation by a free-energy barrier of mainly energetic origin and from the denatured state by a barrier of mainly entropic origin. The general features of the folding pathway seen in all variants of the model four-helix bundles are similar to those observed in the folding of beta-barrel, Greek key proteins; this suggests that many of the qualitative aspects of folding are invariant to the particular native state topology and secondary structure. 相似文献
13.
Kumar A Roach C Hirsh IS Turley S deWalque S Michels PA Hol WG 《Journal of molecular biology》2001,307(1):271-282
A number of helix-rich protein motifs are involved in a variety of critical protein-protein interactions in living cells. One of these is the tetratrico peptide repeat (TPR) motif that is involved, amongst others, in cell cycle regulation, chaperone function and post-translation modifications. So far, these helix-rich TPR motifs have always been observed to be a compact unit of two helices interacting with each other in antiparallel fashion. Here, we describe the structure of the first three TPR-motifs of the peroxin PEX5 from Trypanosoma brucei, the causative agent of sleeping sickness. Peroxins are proteins involved in peroxisome, glycosome and glyoxysome biogenesis. PEX5 is the receptor of the proteins targeted to these organelles by the "peroxisomal targeting signal-1", a C-terminal tripeptide called PTS-1. The first two of the three TPR-motifs of T. brucei PEX5 appear to adopt the canonical antiparallel helix hairpin structure. In contrast, the third TPR motif of PEX5 has a dramatically different conformation in our crystals: the two helices that were supposed to form a hairpin are folded into one single 44 A long continuous helix. Such a conformation has never been observed before for a TPR motif. This raises interesting questions including the potential functional importance of a "jack-knife" conformational change in TPR motifs. 相似文献
14.
DbpA is a DEAD‐box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA‐like catalytic core responsible for double‐helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site‐specific enzyme or region‐specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region‐specific or a site‐specific enzyme. Our data suggest that DbpA is a region‐specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C‐terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double‐helices in its vicinity. The only requirement for a double‐helix to serve as a DbpA substrate is for the double‐helix to be positioned within the catalytic core's grasp. 相似文献
15.
Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function. 相似文献
16.
Dmitriev OY Fillingame RH 《Protein science : a publication of the Protein Society》2007,16(10):2118-2122
We have tested the role of the polar loop of subunit c of the Escherichia coli ATP synthase in stabilizing the hairpin structure of this protein. The structure of the c(32-52) peptide corresponding to the cytoplasmic region of subunit c bound to the dodecylphosphocholine micelles was solved by high-resolution NMR. The region comprising residues 41-47 forms a well-ordered structure rather similar to the conformation of the polar loop region in the solution structure of the full-length subunit c and is flanked by short alpha-helical segments. This result suggests that the rigidity of the polar loop significantly contributes to the stability of the hairpin formed by the two helices of subunit c. This experimental system may be useful for NMR studies of interactions between subunit c and subunits gamma and epsilon, which together form the rotor of the ATP synthase. 相似文献
17.
Probing hairpin structures of small DNAs by nondenaturing polyacrylamide gel electrophoresis 总被引:1,自引:0,他引:1
The influence of temperature on the electrophoretic mobility of small DNAs, capable of forming hairpin structures, is investigated under nondenaturing conditions. Three series of hairpin-forming DNAs containing different numbers of thymidine, deoxyadenosine, and deoxyguanosine residues in their loop, and an identical sequence in the helical region, are analyzed. All show enhanced electrophoretic mobility if they adopt the hairpin conformation. The same quantitative relationship between hairpin formation and increase in electrophoretic mobility is observed for all of the three series. The constancy of this increase suggests a dependence of electrophoretic acceleration on the length of the helical region. A possible application of nondenaturing electrophoresis is monitoring the hairpin/coil transition. Another possible application is the detection of dimers formed by partially self-complementary sequences. This dimer formation is detected for completely complementary DNAs, whereas sequences which might form imperfect double helices, especially those with three bulged-out nucleotides, prefer hairpin formation. The possible applications are experimentally approached and discussed. 相似文献
18.
P Anderson J Monforte R Tritz S Nesbitt J Hearst A Hampel 《Nucleic acids research》1994,22(6):1096-1100
Extensive in vitro mutagenesis studies have been performed on the hairpin ribozyme and substrate in an effort to refine the overall secondary structure of the molecule and provide further insight into what elements are essential for activity. A secondary structure consisting of four helices and five loop regions remains the basic model as originally proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our results suggest that helices 3 and 4 are smaller than previously proposed, consisting of four base pairs and three base pairs respectively. Helix 4 can be extended without loss of activity and loop 3 at the closed end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 consisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made between the new data and previously published mutagenesis and phylogenetic data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage (*) occurring in a preferred 5'(g/c/u)n*guc3' sequence of the substrate. 相似文献
19.
Initiation of DNA replication by DNA polymerases from primers forming a triple helix 总被引:1,自引:1,他引:0
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Rocher C Dalibart R Letellier T Précigoux G Lestienne P 《Nucleic acids research》2001,29(16):3320-3326
Despite extensive studies on oligonucleotide-forming triple helices, which were discovered in 1957, their possible relevance in the initiation of DNA replication remains unknown. Using sequences forming triple helices, we have developed a DNA polymerisation assay by using hairpin DNA templates with a 3′ dideoxynucleotide end and an unpaired 5′-end extension to be replicated. The T7 DNA polymerase successfully elongated nucleotides to the expected size of the template from the primers forming triple helices composed of 9–14 deoxyguanosine-rich residues. The triple helix-forming primer required for this reaction has to be oriented parallel to the homologous sequence of the hairpin DNA template. Substitution of the deoxyguanosine residues by N7 deazadeoxyguanosines in the hairpin of the template prevented primer elongation, suggesting that the formation of a triple helix is a prerequisite for primer elongation. Furthermore, DNA sequencing could be achieved with the hairpin template through partial elongation of the third DNA strand forming primer. The T4 DNA polymerase and the Klenow fragment of DNA polymerase I provided similar DNA elongation to the T7 polymerase–thioredoxin complex. On the basis of published crystallographic data, we show that the third DNA strand primer fits within the catalytic centre of the T7 DNA polymerase, thus underlying this new property of several DNA polymerases which may be relevant to genome rearrangements and to the evolution of the genetic apparatus, namely the DNA structure and replication processes. 相似文献
20.
The spontaneous insertion of proteins into and across membranes: The helical hairpin hypothesis 总被引:1,自引:0,他引:1
We propose that the initial event in the secretion of proteins across membranes and their insertion into membranes is the spontaneous penetration of the hydrophobic portion of the bilayer by a helical hairpin. Energetic considerations of polypeptide structures in a nonpolar, lipid environment compared with an aqueous environment suggest that only α and 310 helices will be observed in the hydrophobic interior of membranes. Insertion of a polypeptide is accomplished by a hairpin structure composed of two helices, which will partition into membranes if the free energy arising from burying hydrophobic helical surfaces exceeds the free energy “cost” of burying potentially charged and hydrogen-bonding groups. We suggest, for example, that the hydrophobic leader peptide found in secreted proteins and in many membrane proteins forms one of these helices and is oriented in the membrane with its N terminus inside. In secreted proteins, the leader functions by pulling polar portions of a protein into the membrane as the second helix of the hairpin. The occurrence of all categories of membrane proteins can be rationalized by the hydrophobic or hydrophilic character of the two helices of the inserted hairpin and, for some integral membrane proteins, by events in which a single terminal helix is inserted. We propose that, because of the distribution of polar and nonpolar sequences in the polypeptide sequence, secretion and the insertion of membrane proteins are spontaneous processes that do not require the participation of additional specific membrane receptors or transport proteins. 相似文献