RESUMO
The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or ß-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.
Assuntos
Proteínas Anticongelantes/química , Proteínas de Artrópodes/química , Glicina/química , Peptídeos/química , Sequência de Aminoácidos , Animais , Proteínas Anticongelantes/genética , Proteínas Anticongelantes/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Cristalografia por Raios X , Expressão Gênica , Glicina/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Peptídeos/metabolismo , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sifonápteros/química , TermodinâmicaRESUMO
Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: ß-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.
Assuntos
Transferência de Energia/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Proteínas Musculares/química , Canais de Sódio/química , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Compostos de Boro/química , Cinética , Elementos da Série dos Lantanídeos/química , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Oócitos/química , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Xenopus/genéticaRESUMO
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Animais , Transferência de Energia , Feminino , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Modelos Moleculares , Oócitos , Conformação Proteica , Domínios Proteicos , Xenopus laevisRESUMO
ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that d-ShK has a near-background IC50 value â¼50,000 times lower than that of the l-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated d-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, d-allo-ShK was made up of d-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing d-allo-Ile and d-allo-Thr along with d-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize d-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both l-allo-ShK and d-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the l-allo-ShK nor the d-allo-ShK polypeptides folded, whereas both l-ShK and d-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported d-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic d-ShK. On the basis of these results, we conclude that the previously reported d-allo-ShK was in fact d-ShK, the true enantiomer of natural l-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of l-ShK toxin.
Assuntos
Venenos de Cnidários/metabolismo , Anêmonas-do-Mar/química , Animais , Venenos de Cnidários/química , Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.3/metabolismo , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Anêmonas-do-Mar/metabolismoRESUMO
Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.
Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalização , Cristalografia por Raios X , Escherichia coli/metabolismo , Humanos , Conformação Molecular , Dados de Sequência Molecular , Mycobacterium tuberculosis/metabolismo , Peptídeos/química , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Ribossomos/química , Estereoisomerismo , Thermus/metabolismoRESUMO
We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-ß-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.
Assuntos
Hipoglicemiantes/síntese química , Insulina Lispro/síntese química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Dissulfetos/química , Fluorenos/química , Hipoglicemiantes/química , Insulina Lispro/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Técnicas de Síntese em Fase SólidaRESUMO
ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.
Assuntos
Venenos de Cnidários/química , Isoleucina/química , Dobramento de Proteína , Treonina/química , Estrutura Molecular , Estabilidade Proteica , EstereoisomerismoRESUMO
As a part of a program aimed towards the study of the dynamics of human insulin-protein dimer formation using two-dimensional infrared spectroscopy, we used total chemical synthesis to prepare stable isotope labeled [(1-(13) C=(18) O)Phe(B24) )] human insulin, via [(1-(13) C=(18) O)Phe(B24) )] ester insulin as a key intermediate product that facilitates folding of the synthetic protein molecule (see preceding article). Here, we describe the crystal structure of the synthetic isotope-labeled ester insulin intermediate and the product synthetic human insulin. Additionally, we present our observations on hexamer formation with these two proteins in the absence of phenol derivatives and/or Zn metal ions. We also describe and discuss the fractional crystallization of quasi-racemic protein mixtures containing each of these two synthetic proteins.
Assuntos
Insulina/química , Proteínas/química , Cristalização , Cristalografia por Raios X , Ésteres , Marcação por Isótopo , Modelos Moleculares , Conformação Proteica , EstereoisomerismoRESUMO
Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site-specific incorporation of stable (13) C=(18) O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis-via a key ester insulin intermediate-of 97 % enriched [(1-(13) C=(18) O)Phe(B24) ] human insulin: stable-isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1-(13) C=(18) O)Phe(B24) ] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red-shifted amideâ I carbonyl band peak at 1595â cm(-1) resulting from the (1-(13) C=(18) O)Phe(B24) backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.
Assuntos
Isótopos de Carbono/química , Insulina/química , Isótopos de Oxigênio/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Sequência de Aminoácidos , Humanos , Dados de Sequência MolecularRESUMO
In this paper, we have used total chemical synthesis of RNase A analogues in order to probe the molecular basis of enzyme catalysis. Our goal was to obligately fill the adenine-binding pocket on the enzyme molecule, and to thus pre-orient the imidazole side chain of His119 in its catalytically productive orientation. Two designed analogues of the RNase A protein molecule that contained an adenine moiety covalently bound to distinct amino acid side chains adjacent to the adenine binding pocket were prepared. A crystal structure of one analogue was determined at 2.3 Å resolution. Kinetic data for RNA transphosporylation and 2',3' cyclic mononucleotide hydrolysis were acquired for the adenine-containing RNase A analogue proteins. As anticipated, the presence of a covalently attached adenine on the enzyme molecule decreased the rate of transphosphorylation and increased the rate of hydrolysis, although the magnitude of the effects was small. This work illustrates the use of total protein synthesis to investigate the chemistry of enzyme catalysis in ways not possible through traditional biochemistry or molecular biology.
Assuntos
Ribonuclease Pancreático/síntese química , Adenina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Hidrólise , Simulação de Acoplamento Molecular , Fosforilação , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismoRESUMO
Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus. Ts3 binds to the domain IV voltage sensor of voltage-gated sodium channels (Nav ) and slows down their fast inactivation. The covalent structure of the Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Herein, we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure-activity studies that enabled us to establish the covalent structure of biologically active Ts3 toxin. We also report the synthesis of the mirror image form of the Ts3 protein molecule, and the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X-ray diffraction.
Assuntos
Venenos de Escorpião/química , Potenciais de Ação , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Venenos de Escorpião/síntese química , Venenos de Escorpião/metabolismo , Escorpiões/metabolismo , Relação Estrutura-AtividadeRESUMO
Misfolding of proinsulin variants in the pancreatic ß-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue Gly(B8) is replaced by Ser(B8). Modular total chemical synthesis was used to prepare the wild-type [Gly(B8)]proinsulin molecule and three analogs: [D-Ala(B8)]proinsulin, [L-Ala(B8)]proinsulin, and the clinical mutant [L-Ser(B8)]proinsulin. The protein diastereomer [D-Ala(B8)]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [L-Ser(B8)]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [L-Ser(B8)]proinsulin did not fold well under the physiological conditions investigated, once folded the [L-Ser(B8)]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding.
Assuntos
Alanina/química , Diabetes Mellitus/metabolismo , Doenças do Recém-Nascido/metabolismo , Proinsulina/síntese química , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Humanos , Recém-Nascido , Proinsulina/química , Espectrometria de Massas por Ionização por Electrospray , EstereoisomerismoRESUMO
Peptide chemistry plays a key role in the synthesis and study of protein molecules and their functions. Modern ligation methods enable the total synthesis of enzymes and the systematic dissection of the chemical basis of enzyme catalysis. Predicted developments in peptide science are described.
Assuntos
Disciplinas das Ciências Biológicas/tendências , Química Analítica/tendências , Peptídeos/química , Peptidomiméticos/química , Processamento de Proteína Pós-Traducional , Disciplinas das Ciências Biológicas/métodos , Catálise , Enzimas/química , Enzimas/farmacologia , Previsões , Humanos , Peptídeos/síntese química , Peptídeos/farmacologia , Peptidomiméticos/síntese química , Peptidomiméticos/farmacologia , Vacinas de Subunidades Antigênicas/síntese química , Vacinas de Subunidades Antigênicas/farmacologia , Vacinas SintéticasRESUMO
Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF(165) to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form of VEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å(2) in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.
Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/síntese química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fases de Leitura Aberta , Estereoisomerismo , Ressonância de Plasmônio de Superfície , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.
Assuntos
Protease de HIV/química , Protease de HIV/síntese química , Modelos Moleculares , Conformação Proteica , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica MolecularRESUMO
Ts1 toxin is a protein found in the venom of the Brazilian scorpion Tityus serrulatus. Ts1 binds to the domain II voltage sensor in the voltage-gated sodium channel Nav and modifies its voltage dependence. In the work reported here, we established an efficient total chemical synthesis of the Ts1 protein using modern chemical ligation methods and demonstrated that it was fully active in modifying the voltage dependence of the rat skeletal muscle voltage-gated sodium channel rNav1.4 expressed in oocytes. Total synthesis combined with click chemistry was used to label the Ts1 protein molecule with the fluorescent dyes Alexa-Fluor 488 and Bodipy. Dye-labeled Ts1 proteins retained their optical properties and bound to and modified the voltage dependence of the sodium channel Nav. Because of the highly specific binding of Ts1 toxin to Nav, successful chemical synthesis and labeling of Ts1 toxin provides an important tool for biophysical studies, histochemical studies, and opto-pharmacological studies of the Nav protein.
Assuntos
Corantes Fluorescentes/química , Venenos de Escorpião/síntese química , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Espectrometria de Massas , Dados de Sequência Molecular , Músculo Esquelético/química , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/farmacologiaRESUMO
CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T-cells and acts as a chemoattractant for monocytes. Originally, CCL1 was identified as a 73 amino acid protein having one N-glycosylation site, and a variant 74 residue non-glycosylated form, Ser-CCL1, has also been described. There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser-CCL1. Here we report the total chemical syntheses of both N-glycosylated and non-glycosylated forms of (Ser-)CCL1, by convergent native chemical ligation. We used an N-glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptide-(α) thioester building block. Chemotaxis assays of these glycoproteins and the corresponding non-glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser-)CCL1 using homogeneous N-glycosylated protein molecules of defined covalent structure.
Assuntos
Quimiocina CCL1/síntese química , Serina/química , Sequência de Aminoácidos , Quimiocina CCL1/química , Glicosilação , Dados de Sequência MolecularRESUMO
The enzyme sortaseâ A is a ligase which catalyzes transpeptidation reactions.1, 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly5 on the peptidoglycan of bacterial cell walls by sortaseâ A.1 The enzyme is an important anti-virulence and anti-infective drug target for resistant strains of Gram-positive bacteria.2 In addition, because sortaseâ A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortaseâ A is a potentially valuable tool for protein science.3 Here we describe the total chemical synthesis of enzymatically active sortaseâ A. The target 148 residue polypeptide chain of sortaseâ AΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size-exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortaseâ A will enable more sophisticated engineering of this important enzyme molecule.
Assuntos
Aminoaciltransferases/síntese química , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/síntese química , Cisteína Endopeptidases/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Catálise , Cromatografia em Gel , Dados de Sequência Molecular , Dobramento de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Our goal was to obtain the X-ray crystal structure of the glycosylated chemokine Ser-CCL1. Glycoproteins can be hard to crystallize because of the heterogeneity of the oligosaccharide (glycan) moiety. We used glycosylated Ser-CCL1 that had been prepared by total chemical synthesis as a homogeneous compound containing an N-linked asialo biantennary nonasaccharide glycan moiety of defined covalent structure. Facile crystal formation occurred from a quasi-racemic mixture consisting of glycosylated L-protein and non-glycosylated-D-protein, while no crystals were obtained from the glycosylated L-protein alone. The structure was solved at a resolution of 2.6-2.1â Å. However, the glycan moiety was disordered: only the N-linked GlcNAc sugar was well-defined in the electron density map. A racemic mixture of the protein enantiomers L-Ser-CCL1 and D-Ser-CCL1 was also crystallized, and the structure of the true racemate was solved at a resolution of 2.7-2.15â Å. Superimposition of the structures of the protein moieties of L-Ser-CCL1 and glycosylated-L-Ser-CCL1 revealed there was no significant alteration of the protein structure by N-glycosylation.
Assuntos
Quimiocina CCL1/química , Serina/química , Glicosilação , EstereoisomerismoRESUMO
In the course of an attempted total chemical synthesis of the ant insulin-like peptide-2 (ILP2) protein molecule, specific cleavage of a backbone peptide bond in a branched ester-linked polypeptide chain with concomitant peptide splicing was observed. The side reaction was investigated in model compounds. Here, we postulate a chemical mechanism for this novel polypeptide backbone cleavage reaction as a chemical counterpart to the resolution step of biochemical intein-mediated protein splicing.