RESUMO
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs (AA-tRNAs) to catalyse cyclodipeptide formation in a ping-pong mechanism. Despite intense studies of these enzymes in past years, the tRNA regions of the two substrates required for CDPS activity are poorly documented, mainly because of two limitations. First, previously studied CDPSs use two identical AA-tRNAs to produce homocyclodipeptides, thus preventing the discriminative study of the binding of the two substrates. Second, the range of tRNA analogues that can be aminoacylated by aminoacyl-tRNA synthetases is limited. To overcome the limitations, we studied a new model CDPS that uses two different AA-tRNAs to produce an heterocyclodipeptide. We also developed a production pipeline for the production of purified shortened AA-tRNA analogues (AA-minitRNAs). This method combines the use of flexizymes to aminoacylate a diversity of minitRNAs and their subsequent purifications by anion-exchange chromatography. Finally, we were able to show that aminoacylated molecules mimicking the entire acceptor arms of tRNAs were as effective a substrate as entire AA-tRNAs, thereby demonstrating that the acceptor arms of the two substrates are the only parts of the tRNAs required for CDPS activity. The method developed in this study should greatly facilitate future investigations of the specificity of CDPSs and of other AA-tRNAs-utilizing enzymes.
Assuntos
Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Ensaios Enzimáticos , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacilação de RNA de TransferênciaRESUMO
Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNAPhe to the CDPS from Candidatus Glomeribacter gigasporarum (Cglo-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the Cglo-CDPS:Phe-tRNAPhe complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS). The data show that the major groove of the acceptor stem of the aa-tRNA interacts with the enzyme through the basic ß2 and ß7 strands of CDPSs belonging to the XYP subfamily. A bending of the CCA extremity enables the amino acid moiety to be positioned in the P1 pocket while the terminal A76 adenosine occupies the P2 pocket. Such a positioning indicates that the present structure illustrates the binding of the first aa-tRNA. In cells, CDPSs and the elongation factor EF-Tu share aminoacylated tRNAs as substrates. The present study shows that CDPSs and EF-Tu interact with opposite sides of tRNA. This may explain how CDPSs hijack aa-tRNAs from canonical ribosomal protein synthesis.
Assuntos
Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Burkholderiaceae/efeitos dos fármacos , Burkholderiaceae/genética , Cromatografia em Gel , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(L-isoleucyl-L-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.
Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Genes Bacterianos/genética , Família Multigênica , Streptomyces/genética , Antibacterianos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Dicetopiperazinas/química , Hidroxilação , Modelos Químicos , Estrutura Molecular , Mutação , Streptomyces/metabolismoRESUMO
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue.
Assuntos
Peptídeo Sintases/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de Proteína , Relação Estrutura-AtividadeRESUMO
Cyclodipeptide synthases (CDPSs) use as substrates two amino acids activated as aminoacyl-tRNAs to synthesize cyclodipeptides in secondary metabolites biosynthetic pathways. Since the first description of a CDPS in 2002, the number of putative CDPSs in databases has increased exponentially, reaching around 800 in June 2017. They are likely to be involved in numerous biosynthetic pathways but the diversity of their products is still under-explored. Here, we describe the activity of 32 new CDPSs, bringing the number of experimentally characterized CDPSs to about 100. We detect 16 new cyclodipeptides, one of which containing an arginine which has never been observed previously. This brings to 75 the number of cyclodipeptides formed by CDPSs out of the possible 210 natural ones. We also identify several consensus sequences related to the synthesis of a specific cyclodipeptide, improving the predictive model of CDPS specificity. The improved prediction method enables to propose the main product synthesized for about 80% of the CDPS sequences available in databases and opens the way for the deciphering of CDPS-dependent pathways. Analysis of phylum distribution and predicted activity for all CDPSs identified in databases shows that the experimentally characterized set is representative of the whole family. Our work also demonstrates that some cyclodipeptides, precursors of diketopiperazines with interesting pharmacological properties and previously described as being synthesized by fungal non-ribosomal peptide synthetases, can also be produced by CDPSs in bacteria.
RESUMO
The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5-diketopiperazines (2,5-DKPs) have been widely developed by medicinal chemists, but their biological production is yet to be exploited. We introduce an inâ vivo method for incorporating non-canonical amino acids (ncAAs) into 2,5-DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5-DKP biosynthetic pathways. CDPSs use aminoacyl-tRNAs as substrates. We exploited the natural ability of aminoacyl-tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non-canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5-DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide-tailoring enzymes found in 2,5-DKP biosynthetic pathways.
Assuntos
Aminoácidos/metabolismo , Dicetopiperazinas/metabolismo , Peptídeo Sintases/metabolismo , Aminoácidos/química , Dicetopiperazinas/química , Conformação MolecularRESUMO
A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
RESUMO
Hfq is a bacterial RNA binding protein that carries out several roles in genetic expression regulation, mainly at the post-transcriptional level. Previous studies have shown its importance in growth and virulence of bacteria. Here, we provide the direct observation of its ability to interact with membranes. This was established by co-sedimentation assay, cryo-transmission electron (cryo-TEM) and atomic force (AFM) microscopies. Furthermore, our results suggest a role for its C-terminus amyloidogenic domain in membrane disruption. Precisely, AFM images of lipid bilayers in contact with Hfq C-terminus fibrils show the emergence of holes with a size dependent on the time of interaction. Cryo-TEM observations also show that liposomes are in contact with clusters of fibrils, with occasional deformation of the vesicles and afterward the apparition of a multitude of tiny vesicles in the proximity of the fibrils, suggesting peptide-induced breakage of the liposomes. Finally, circular dichroism spectroscopy demonstrated a change in the secondary structure of Hfq C-terminus upon interaction with liposomes. Altogether, these results show an unexpected property of Hfq and suggest a possible new role for the protein, exporting sRNA outside of the bacterial cell.
RESUMO
Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.
Assuntos
Proteínas de Bactérias/química , Dipeptídeos/química , Proteínas Fúngicas/química , Peptídeo Sintases/química , Peptídeos Cíclicos/química , Motivos de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biologia Computacional , Ciclização , Bases de Dados Genéticas , Dipeptídeos/biossíntese , Dipeptídeos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Expressão Gênica , Dados de Sequência Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/biossíntese , Peptídeo Sintases/genética , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/genética , Filogenia , Estrutura Terciária de Proteína , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por SubstratoRESUMO
Cyclodipeptide synthases form cyclodipeptides from two aminoacyl transfer RNAs. They use a ping-pong mechanism that begins with transfer of the aminoacyl moiety of the first aminoacyl tRNA onto a conserved serine, yielding an aminoacyl enzyme. Combining X-ray crystallography, site-directed mutagenesis and affinity labelling of the cyclodipeptide synthase AlbC, we demonstrate that the covalent intermediate reacts with the aminoacyl moiety of the second aminoacyl tRNA, forming a dipeptidyl enzyme, and identify the aminoacyl-binding sites of the aminoacyl tRNAs.
Assuntos
Proteínas de Bactérias/química , Biossíntese Peptídica , Peptídeo Sintases/química , Ribossomos/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Conformação Molecular , Mutagênese Sítio-Dirigida , RNA de Transferência/química , Serina/química , Streptomyces/químicaRESUMO
Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNA(Phe) is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix α4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNA(Leu) isoacceptors as a second substrate. We show that the G(1)-C(72) pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop α6-α7 or D205 located in the loop ß6-α8 affected Leu-tRNA(Leu) isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites.
Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Sítios de Ligação , Peptídeo Sintases/química , Aminoacil-RNA de Transferência/química , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , RNA de Transferência de Fenilalanina/química , RNA de Transferência de Fenilalanina/metabolismo , Especificidade por SubstratoRESUMO
We review here work on the biosynthesis of diketopiperazines (DKPs), a large class of natural products with noteworthy biological activities, focusing on the biosynthetic pathways involving cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. Distinct from nonribosomal peptide synthetases (NRPSs), the other family of enzymes synthesizing DKPs, CDPSs bridge the primary and secondary metabolic pathways by hijacking aminoacyl-tRNAs to produce DKPs. This review includes a comprehensive description of the state of the art for CDPS-dependent pathways, and highlights the ways in which this knowledge could be used to increase the diversity of natural DKPs by pathway engineering.
Assuntos
Produtos Biológicos/síntese química , Dicetopiperazinas/síntese química , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Sequência de Aminoácidos , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Dicetopiperazinas/química , Dicetopiperazinas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Conformação ProteicaRESUMO
Cyclodipeptide synthases (CDPSs) are small enzymes structurally related to class-I aminoacyl-tRNA synthetases (aaRSs). They divert aminoacylated tRNAs from their canonical role in ribosomal protein synthesis, for cyclodipeptide formation. All the CDPSs experimentally characterized to date are bacterial. We show here that a predicted CDPS from the sea anemone Nematostella vectensis is an active CDPS catalyzing the formation of various cyclodipeptides, preferentially containing tryptophan. Our findings demonstrate that eukaryotes encode active CDPSs and suggest that all CDPSs have a similar aminoacyl-tRNA synthetase-like architecture and ping-pong mechanism. They also raise questions about the biological roles of the cyclodipeptides produced in bacteria and eukaryotes.
Assuntos
Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/biossíntese , Anêmonas-do-Mar/enzimologia , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/química , Peptídeo Sintases/genética , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.
Assuntos
Proteínas de Bactérias/química , Dipeptídeos/biossíntese , Peptídeo Sintases/química , Peptídeos Cíclicos/biossíntese , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cristalografia , Modelos Moleculares , Dados de Sequência Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologiaRESUMO
The core light-harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n-octyl-beta-D-glucopyranoside into smaller subunit forms, exhibiting a dramatic blue-shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of n-octyl-beta-D-glucopyranoside, we have previously shown that the B820 form is not only constituted by the alphabeta heterodimer alone, but that it exists in an equilibrium between the alphabeta heterodimer and beta(2) homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoretical model for alphabeta and beta(2), we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non-covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.
Assuntos
Proteínas de Bactérias/química , Entropia , Complexos de Proteínas Captadores de Luz/química , Rhodospirillum rubrum/enzimologia , Dimerização , Estabilidade Enzimática , Glucosídeos/química , Modelos Moleculares , Peptídeos/química , Conformação ProteicaRESUMO
Light harvesting complexes 2 (LH2) are the peripheral antenna proteins in the bacterial photosynthetic apparatus and are built of alpha/beta-heterodimers containing three bacteriochlorophylls and two carotenoids each. Previously, we have found in 2D-crystals that the complexes could be inserted within the membrane with a tilt with respect to the membrane plane (Rhodobacter sphaeroides) or without tilt (Rubrivivax gelatinosus). To investigate whether the tilted insertion represents the native state or if it is due to specific 2D-crystal contacts, we have used atomic force microscopy to investigate LH2 from Rhodopseudomonas acidophila reconstituted at different lipid to protein ratios. High-resolution topographs could be acquired of two types of 2D-crystals or of densely packed membranes. Interestingly, in type 2 2D-crystals and in non-crystalline densely packed membranes, cylinders are integrated with their symmetry axis normal to the membrane plane, while in type 1 2D-crystals LH2 cylinders are integrated with a tilt of approximately 4 degrees with respect to the membrane plane. Therefore, we present strong evidence that the tilt of LH2 does not represent the native membrane state and is due to protein-protein contacts in specific 2D-crystals.
Assuntos
Membrana Celular/química , Complexos de Proteínas Captadores de Luz/química , Microscopia de Força Atômica , Rodopseudomonas/química , Membrana Celular/metabolismo , Cristalização , Complexos de Proteínas Captadores de Luz/metabolismo , LipídeosRESUMO
The Hfq (Host factor 1) polypeptide is a nucleic acid binding protein involved in the synthesis of many polypeptides. Hfq particularly affects the translation and the stability of several RNAs. In an earlier study, the use of fold recognition methods allowed us to detect a relationship between Escherichia coli Hfq and the Sm topology. This topology was further validated by a series of biophysical studies and the Hfq structure was modelled on an Sm protein. Hfq forms a beta-sheet ring-shaped hexamer. As our previous study predicted a large number of alternative conformations for the C-terminal region, we have determined whether the last 19 C-terminal residues are necessary for protein function. We find that the C-terminal truncated protein is fully capable of binding a polyadenylated RNA (K(d) of 120 pm vs. 50 pm for full-length Hfq). This result shows that the functional core of E. coli Hfq resides in residues 1-70 and confirms previous genetic studies. Using equilibrium unfolding studies, however, we find that full-length Hfq is 1.8 kcal x mol(-1) more stable than its truncated variant. Electron microscopy analysis of both truncated and full-length proteins indicates a structural rearrangement between the subunits upon truncation. This conformational change is coupled to a reduction in beta-strand content, as determined by Fourier transform infra-red. On the basis of these results, we propose that the C-terminal domain could protect the interface between the subunits and stabilize the hexameric Hfq structure. The origin of this C-terminal domain is also discussed.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Processamento de Imagem Assistida por Computador , Cinética , Espectrometria de Massas , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , Ureia/farmacologiaRESUMO
The effect of partial digestion by trypsin and GluC protease on the association of the membrane polypeptides of LH1 from Rhodospirillum (Rsp.) rubrum was studied. Trypsin and GluC protease treatments of LH1 result in the cleavage of the first three amino acids from the alpha polypeptide and of the first 18 amino acids from the beta polypeptide, respectively, without any noticeable reorganization of their secondary structure, as measured by attenuated total reflectance Fourier transform IR spectroscopy. However, the enthalpy variation accompanying dimer formation was dramatically reduced by the protease attacks by as much as 80%. Our results show that the alphabeta heterodimer is mainly stabilized by hydrophobic interactions which involve the amino-terminal extensions of the participating polypeptides. Using the close homology between the polypeptides of Rsp. rubrum LH1 and that of Rsp. molischianum LH2, whose structure is known, a structural model for these "hydrophobic pockets" lying close to the membrane interface is proposed.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrólise , Dados de Sequência Molecular , Concentração Osmolar , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rhodobacter sphaeroides , Rodopseudomonas , Rhodospirillum rubrum , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , Tripsina/químicaRESUMO
In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution in situ. Here we present high-resolution AFM of the photosynthetic core complex in native Rhodopseudomonas viridis membranes. Topographs at 10-A lateral and approximately 1-A vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 light-harvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex.
Assuntos
Nanotecnologia , Complexo de Proteínas do Centro de Reação Fotossintética/ultraestrutura , Rodopseudomonas/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica , Rodopseudomonas/ultraestruturaRESUMO
Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.