RESUMO
Peptides have been instrumental in the development of solid-state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid-state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple-resonance methods are described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples are presented.
Assuntos
Espectroscopia de Ressonância Magnética/instrumentação , Espectroscopia de Ressonância Magnética/métodos , Peptídeos/químicaRESUMO
Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Chaperonas Moleculares/química , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA. The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex. Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG. A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation. Several recent reports show that functions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA. We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA. Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant. Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Sinais Direcionadores de Proteínas , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Biotina/metabolismo , Proteínas de Transporte/química , Quimotripsina/metabolismo , Dicroísmo Circular , Densitometria , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
The signal recognition particle (SRP) is an RNA-protein complex that directs ribosomes to the rough endoplasmic reticulum membrane by binding to targeting signals found on the nascent chain of proteins destined for export to the endoplasmic reticulum. We found evidence from studies with fragments of the protein component of the Escherichia coli SRP that a long hydrophobic loop (the so-called "finger loop") is detrimental to the stability of its signal peptide-binding domain, the M domain. This hydrophobic loop is highly conserved and thus may have a critical role in the function of the SRP. Given our previously reported evidence that 4.5 S RNA stabilizes the tertiary fold of the M domain (Zheng, N., and Gierasch, L. M. (1997) Mol. Cell 1, 79-87), we now propose that the functional requirement for 4.5 S RNA resides in its ability to counteract the destabilizing influence of the finger loop.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/metabolismo , RNA Ribossômico/química , Partícula de Reconhecimento de Sinal/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático Rugoso/metabolismo , Humanos , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Bacteriano , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Investigators have recently turned to studies of protein families to shed light on the mechanism of protein folding. In small proteins for which detailed analysis has been performed, recent studies show that transition-state structure is generally conserved. The number and structures of populated folding intermediates have been found to vary in homologous families of larger (greater than 100-residue) proteins, reflecting a balance of local and global interactions.
Assuntos
Modelos Químicos , Família Multigênica , Dobramento de Proteína , Sequência de Aminoácidos , Simulação por Computador , Sequência Conservada , Evolução Molecular , Cinética , Modelos MolecularesRESUMO
N-terminal signal sequences can direct nascent protein chains to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes by interacting with the signal recognition particle. In this study, we show that isolated peptides corresponding to several bacterial signal sequences inhibit the GTPase activity of the Escherichia coli signal recognition particle, as previously reported (Miller, J. D., Bernstein, H. D., and Walter, P. (1994) Nature 367, 657-659), but not by the direct mechanism proposed. Instead, isolated signal peptides bind nonspecifically to the RNA component and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity. Surprisingly, only "functional" peptide sequences aggregate RNA; the peptides in general use as "nonfunctional" negative controls (e.g. those with deletions or charged substitutions within the hydrophobic core), are sufficiently different in physical character that they do not aggregate RNA and thus have no effect on the GTPase activity of the signal recognition particle. We propose that the reported effect of functional signal peptides on the GTPase activity of the signal recognition particle is an artifact of the high peptide concentrations and low salt conditions used in these in vitro studies and that signal sequences at the N terminus of nascent chains in vivo do not exhibit this activity.
Assuntos
GTP Fosfo-Hidrolases/antagonistas & inibidores , Sinais Direcionadores de Proteínas , RNA/metabolismo , Partícula de Reconhecimento de Sinal , Ligação Proteica , Eletricidade EstáticaRESUMO
To explore the ways that proline residues may influence the conformational options of a polypeptide backbone, we have characterized Pro-->Ala mutants of cellular retinoic acid-binding protein I (CRABP I). While all three Xaa-Pro bonds are in the trans conformation in the native protein and the equilibrium stability of each mutant is similar to that of the parent protein, each has distinct effects on folding and unfolding kinetics. The mutation of Pro105 does not alter the kinetics of folding of CRABP I, which indicates that the flexible loop containing this residue is passive in the folding process. By contrast, replacement of Pro85 by Ala abolishes the observable slow phase of folding, revealing that correct configuration of the 84-85 peptide bond is prerequisite to productive folding. Substitution of Pro39 by Ala yields a protein that folds and unfolds more slowly. Removal of the conformational constraint imposed by the proline ring likely raises the transition state barrier by increasing the entropic cost of narrowing the conformational ensemble. Additionally, the Pro-->Ala mutation removes a helix-termination signal that is important for efficient folding to the native state.
Assuntos
Prolina/química , Dicroísmo Circular , Escherichia coli/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Mutação , Prolina/genética , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Temperatura , Termodinâmica , Ureia/farmacologiaRESUMO
Cellular retinoic acid binding protein I (CRABPI) belongs to the family of intracellular lipid binding proteins (iLBPs), all of which bind a hydrophobic ligand within an internal cavity. The structures of several iLBPs reveal minimal structural differences between the apo (ligand-free) and holo (ligand-bound) forms, suggesting that dynamics must play an important role in the ligand recognition and binding processes. Here, a variety of nuclear magnetic resonance (NMR) spectroscopy methods were used to systematically study the dynamics of both apo and holo CRABPI at various time scales. Translational and rotational diffusion constant measurements were used to study the overall motions of the proteins. Both apo and holo forms of CRABPI tend to self-associate at high (1.2 mM) concentrations, while at low concentrations (0.2 mM), they are predominantly monomeric. Rapid amide exchange rate and laboratory frame relaxation rate measurements at two spectrometer field strengths (500 and 600 MHz) were used to probe the internal motions of the individual residues. Several residues in the apo form, notably within the ligand recognition region, exhibit millisecond time scale motions that are significantly arrested in the holo form. In contrast, no significant differences in the high-frequency motions were observed between the two forms. These results provide direct experimental evidence for dynamics-induced ligand recognition and binding at a specifically defined time scale. They also exemplify the importance of dynamics in providing a more comprehensive understanding of how a protein functions.
Assuntos
Receptores do Ácido Retinoico/química , Apoproteínas/química , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Prótons , Relação Estrutura-Atividade , TermodinâmicaRESUMO
How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Polarização de Fluorescência , Proteínas de Choque Térmico HSP70/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The unfolding dynamics of cellular retinoic acid-binding protein I (CRABP I), an 18 kDa predominantly beta-sheet protein, were studied by monitoring the hydrogen-deuterium (H-D) exchange reaction under various solution conditions. A bimodal charge state distribution was observed when a denaturing agent was added to the protein aqueous solution. These two populations exhibit different kinetics of H-D exchange, with the high charge state ions undergoing very rapid isotope exchange, while the low charge state protein ions exchange cooperatively but at much slower rates. Transiently populated intermediate states were detected indirectly using hydrogen exchange measurement in aqueous solution at various pHs. At pH 2.5 and room temperature, three distinct populations of CRABP I ions exist over an extended period of time, each corresponding to a specific degree of backbone amide hydrogen atom protection. Mass spectral data are complementary to hydrogen exchange measurements by NMR, since the former samples a much faster time-scale of dynamic events in solution.
Assuntos
Receptores do Ácido Retinoico/química , Deutério , Hidrogênio , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Soluções , ÁguaRESUMO
The molecular chaperonins are essential proteins involved in protein folding, complex assembly, and polypeptide translocation. While there is abundant structural information about the machinery and the mechanistic details of its action are well studied, it is yet unresolved how chaperonins recognize a large number of structurally unrelated polypeptides in their unfolded or partially folded forms. To determine the nature of chaperonin-substrate recognition, we have characterized by NMR methods the interactions of GroEL with synthetic peptides that mimic segments of unfolded proteins. In previous work, we found using transferred nuclear Overhauser effect (trNOE) analysis that two 13 amino acid peptides bound GroEL in an amphipathic alpha-helical conformation. By extending the study to a variety of peptides with differing sequence motifs, we have observed that peptides can adopt conformations other than alpha-helix when bound to GroEL. Furthermore, peptides of the same composition exhibited significantly different affinities for GroEL as manifested by the magnitude of trNOEs. Binding to GroEL correlates well with the ability of the peptide to cluster hydrophobic residues on one face of the peptide, as determined by the retention time on reversed-phase (RP) HPLC. We conclude that the molecular basis of GroEL-substrate recognition is the presentation of a hydrophobic surface by an incompletely folded polypeptide and that many backbone conformations can be accommodated.
Assuntos
Chaperonina 60/metabolismo , Sequência de Aminoácidos , Chaperonina 60/química , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
In Escherichia coli, DnaK is essential for the replication of bacteriophage lambda DNA; this in vivo activity provides the basis of a screen for mutations affecting DnaK function. Mn PCR was used to introduce mutations into residues 405-468 of the C-terminal polypeptide-binding domain of DnaK. These mutant proteins were screened for the ability to propagate bacteriophage lambda in the background of a dnaK deficient cell line, BB1553. This initial screen identified several proteins which were mutant at multiple positions. The multiple mutants were further dissected into single mutants which remained negative for lambda propagation. Four of these single-site mutants were purified and assayed for biochemical functionality. Two single-site mutations, F426S and S427P, are localized in the peptide binding site and display weakened peptide binding affinity. This indicates that the crystallographically determined peptide binding site is also critical for in vivo lambda replication. Two other mutations, K414I and N451K, are located at the edge of the beta-sandwich domain near alpha-helix A. The K414I mutant binds peptide moderately well, yet displays defects in allosteric functions, including peptide-stimulated ATPase activity, ATP-induced changes in tryptophan fluorescence, ATP-induced peptide release, and elevated ATPase activity. The K414 position is close in tertiary structure to the linker region to the ATPase domain and reflects a specific area of the peptide-binding domain which is necessary for interdomain coupling. The mutant N451K displays defects in both peptide binding and allosteric interaction.
Assuntos
Sítios de Ligação/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Choque Térmico HSP70/química , Chaperonas Moleculares/química , Adenosina Trifosfatases/metabolismo , Regulação Alostérica/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófago lambda/genética , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Cristalografia por Raios X , Escherichia coli/química , Polarização de Fluorescência , Proteínas de Choque Térmico HSP70/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Mutação/genética , Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
BACKGROUND: Cellular retinoic acid binding protein I (CRABPI) is a small, predominantly beta-sheet protein with a simple architecture and no disulfides or cofactors. Folding of mutants containing only one of the three native tryptophans has been examined using stopped-flow fluorescence and circular dichroism at multiple wavelengths. RESULTS: Within 10 ms, the tryptophan fluorescence of all three mutants shows a blue shift, and stopped-flow circular dichroism shows significant secondary structure content. The local environment of Trp7, a completely buried residue located near the intersection of the N and C termini, develops on a 100 ms time scale. Spectral signatures of the other two tryptophan residues (87 and 109) become native-like in a 1 s kinetic phase. CONCLUSIONS: Formation of the native beta structure of CRABPI is initiated by rapid hydrophobic collapse, during which local segments of chain adopt significant secondary structure. Subsequently, transient yet specific interactions of amino acid residues restrict the arrangement of the chain topology and initiate long-range associations such as the docking of the N and C termini. The development of native tertiary environments, including the specific packing of the beta-sheet sidechains, occurs in a final, highly cooperative step simultaneous with stable interstrand hydrogen bonding.
Assuntos
Receptores do Ácido Retinoico/química , Triptofano/química , Dicroísmo Circular , Fluorescência , Cinética , Sondas Moleculares , Estrutura Secundária de ProteínaRESUMO
The Clp/Hsp100 molecular chaperones are unusual in their ability to tease apart protein aggregates and complexes. Recent results make a good case that these chaperones bind substrates via PDZ-like domains; this may reflect a general strategy for manipulating the] assembly state of substrate proteins.
Assuntos
Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Proteínas de Protozoários/química , Serina Endopeptidases/química , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/metabolismo , Endopeptidase Clp , Proteínas de Escherichia coli , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiologia , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/fisiologia , Serina Endopeptidases/metabolismo , Serina Endopeptidases/fisiologia , Especificidade por SubstratoRESUMO
Previously the hexapeptide motif FXNPXY807 in the cytoplasmic tail of the LDL receptor was shown to be essential for clustering in clathrin-coated pits. We used nuclear magnetic resonance line-broadening and transferred nuclear Overhauser effect measurements to identify the molecule in the clathrin lattice that interacts with this hexapeptide, and determined the structure of the bound motif. The wild-type peptide bound in a single conformation with a reverse turn at residues NPVY. Tyr807Ser, a peptide that harbors a mutation that disrupts receptor clustering, displayed markedly reduced interactions. Clustering motif peptides interacted with clathrin cages assembled in the presence or absence of AP2, with recombinant clathrin terminal domains, but not with clathrin hubs. The identification of terminal domains as the primary site of interaction for FXNPXY807 suggests that adaptor molecules are not required for receptor-mediated endocytosis of LDL, and that at least two different tyrosine-based internalization motifs exist for clustering receptors in coated pits.
Assuntos
Clatrina/metabolismo , Conformação Proteica , Receptores de LDL/química , Receptores de LDL/metabolismo , Complexo 2 de Proteínas Adaptadoras , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Animais , Sítios de Ligação , Bovinos , Clatrina/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The time course of folding of a small beta-sheet protein reveals formation of a central ligand binding cavity before the consolidation of the native hydrogen bonding network. These results suggest that side chain interactions and not stable hydrogen bonding determine the beta-sheet architecture and play crucial roles in the overall chain topology.
Assuntos
Dobramento de Proteína , Receptores do Ácido Retinoico/química , Ligação de Hidrogênio , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: As an approach to understanding the role of local sequence in determining protein tertiary structure, we have examined the conformation of a 23-residue peptide fragment corresponding to the structurally conserved helix-Schellman motif-helix (H-Sm-H) domain (residue 10-32) of cellular retinoic acid binding protein, along with variants designed to probe the contributions of the helix-terminating Gly23 and the hydrophobic interactions between Leu 19 and Val24 in stabilizing the Schellman motif and hence helix termination. RESULTS: In aqueous solution, NMR data for the H-Sm-H peptide show that it samples a largely helical conformation with a break in the helix at the point of the turn in the protein. The data also establish the presence of local hydrophobic interactions and intramolecular hydrogen bonds characteristic of a Schellman motif. Absence of helix termination in trifluoroethanol, a solvent known to disrupt hydrophobic interactions, along with an analysis of H alpha chemical shifts and NOEs in the variant peptides, suggest a major role for glycine in terminating the helix, with local hydrophobic interactions further stabilizing the Schellman motif. CONCLUSIONS: The presence of a Schellman motif in this isolated fragment in water is governed by local interactions and specifies the interspatial arrangement of the helices. This observation underlines the structure predictive value of folding motifs. As proposed for a Schellman motif, helix termination in this fragment is dictated by the local distribution of polar/apolar residues, which is reminiscent of the binary code for protein folding.
Assuntos
Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The E. coli protein, Fth, binds to 4.5S RNA through its M domain to form the signal recognition particle (SRP). The other domain of Fth (NG) is a GTPase, which binds and is coordinately regulated by its receptor, FtsY. We find that the helical M domain is inherently flexible. Binding of 4.5S RNA to Fth stabilizes the M domain yet has little apparent effect on the binding of signal peptides. However, in the absence of the RNA, signal peptide binding results in a global destabilization of Fth, which is prevented by binding of 4.5S RNA. Signal peptide binding to isolated NG domain also causes a pronounced destabilization, implicating the NG domain in direct recognition of signal peptide.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli , Escherichia coli/química , RNA Bacteriano/metabolismo , Partícula de Reconhecimento de Sinal/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismoRESUMO
The orientations of helical peptides in membrane bilayers provide important structural information that is directly relevant to their functional roles, both alone and within the context of larger membrane proteins. The orientations can be readily determined with solid state NMR experiments on samples of 15N-labeled peptides in lipid bilayers aligned between glass plates. The observed 15N chemical shift frequencies can be directly interpreted to indicate whether the peptide's helix axis has a trans-membrane or an in-plane orientation. In order to distinguish between these possibilities on the basis of a single spectral parameter, e.g. the easily measured 15N chemical shift frequency, it is necessary to demonstrate that the secondary structure of the peptide is helical, generally by solution NMR spectroscopy of the same peptide in micelle samples, and that it is immobile in bilayers, generally from solid state NMR spectra of unoriented samples. Six different 20-30 residue peptides are shown to have orientations that fall into the categories of trans-membrane or in-plane helices. A model hydrophobic peptide was found to be trans-membrane, several different amphipathic helical peptides were found to have either trans-membrane or in-plane orientations, and a leader or signal peptide, generally regarded as hydrophobic, was found to have a significant population with an in-plane orientation.