RESUMEN
IR spectroscopy of gas-phase ions is proposed to resolve positional isomers of sulfated carbohydrates. Mass spectrometric fingerprints and gas-phase vibrational spectra in the near and mid-IR regions were obtained for sulfated monosaccharides, yielding unambiguous signatures of sulfated isomers. We report the first systematic exploration of the biologically relevant but notoriously challenging deprotonated state in the near IR region. Remarkably, anions displayed very atypical vibrational profiles, which challenge the well-established DFT (Density Functionnal Theory) modeling. The proposed approach was used to elucidate the sulfate patterns in glycosaminoglycans, a ubiquitous class of mammalian carbohydrates, which is regarded as a major challenge in carbohydrate structural analysis. Isomeric glycosaminoglycan disaccharides from heparin and chondroitin sources were resolved, highlighting the potential of infrared multiple photon dissociation spectroscopy as a novel structural tool for carbohydrates.
Asunto(s)
Carbohidratos/química , Fotones , Sulfatos/química , Rayos Infrarrojos , Teoría Cuántica , Espectrofotometría InfrarrojaRESUMEN
BACKGROUND: Diverse varieties of often heterogeneous glycans are ubiquitous in nature. They play critical roles in recognition events, act as energy stores and provide structural stability at both molecular and cellular levels. Technologies capable of fully elucidating the structures of glycans are far behind the other '-omic' fields. Liquid chromatography (LC) and mass spectrometry (MS) are currently the most useful techniques for high-throughput analysis of glycans. However, these techniques do not provide full unambiguous structural information and instead the gap in full sequence assignment is frequently filled by a priori knowledge of the biosynthetic pathways and the assumption that these pathways are highly conserved. SCOPE OF THE REVIEW: This comprehensive review details the rise of the emerging analytical technique ion mobility spectrometry (IMS) (coupled to MS) to facilitate the determination of three-dimensional shape: the separation and characterization of isobaric glycans, glyco(peptides/proteins), glycolipids, glycosaminoglycans and other polysaccharides; localization of sites of glycosylation; or interpretation of the conformational change to proteins upon glycan binding. MAJOR CONCLUSIONS: IMS is a highly promising new analytical route, able to provide rapid isomeric separation (ms timescale) of either precursor or product ions facilitating MS characterization. This additional separation also enables the deconvolution of carbohydrate MS(/MS) information from contaminating ions, improving sensitivity and reducing chemical noise. Derivation of collision cross sections (CCS) from IM-MS(/MS) data and subsequent calculations validate putative structures of carbohydrates from ab initio derived candidates. IM-MS has demonstrated that amounts of specific glycan isomers vary between disease states, which would be challenging to detect using standard analytical approaches. GENERAL SIGNIFICANCE: IM-MS is a promising technique that fills an important gap within the Glycomics toolbox, namely identifying and differentiating the three-dimensional structure of chemically similar carbohydrates and glycoconjugates. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.
Asunto(s)
Espectrometría de Masas/métodos , Polisacáridos/análisis , Animales , HumanosRESUMEN
Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.
Asunto(s)
Carbohidratos/química , Glicopéptidos/química , Espectrometría de Masas/métodos , Polisacáridos/química , Secuencia de Aminoácidos , Glicosiltransferasas/química , Humanos , Datos de Secuencia MolecularRESUMEN
Glycerol derivatives are a class of compounds, which are easy and inexpensive to produce with potent anti-malarial activities against blood stages of Plasmodium falciparum in vitro. In the present study, one of these compounds, termed 1t, which had the lowest IC(50) values, was assessed in a murine malarial model. Nuclear magnetic resonance imaging and Balb/c mice infected with Plasmodium berghei ANKA strain were treated in a 4-day suppressive test. Mice received a once-daily intraperitoneal administration of 50 mg/Kg of the drug for 4 days. Although no parasitaemia clearance was reached, a slower parasite proliferation and a slightly longer survival time compared with the placebo group were observed.
Asunto(s)
Amino Alcoholes/uso terapéutico , Antimaláricos/uso terapéutico , Malaria/tratamiento farmacológico , Plasmodium berghei/efectos de los fármacos , Amino Alcoholes/administración & dosificación , Amino Alcoholes/farmacología , Animales , Antimaláricos/administración & dosificación , Antimaláricos/farmacología , Femenino , Concentración 50 Inhibidora , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos BALB C , Parasitemia/tratamiento farmacológico , Análisis de SupervivenciaRESUMEN
The phospholipid metabolism of Plasmodium falciparum-infected erythrocytes has been shown to be an effective pharmacological target for novel chemotherapy. Thirty-seven monoquaternary ammonium derivatives analogous to choline were screened for their potential antiprotozoal activity against P. falciparum and Leishmania braziliensis. Twenty-three compounds inhibited chloroquine resistant and sensitive P. falciparum strains with inhibitory concentrations ranging from 0.001 microM to 47 microM. Among the inhibitors were six compounds with nanomolar activity containing at least one ethyl group in the polar head and a hydrophobic alkyl chain with 10 to 14 methylene groups. Four compounds also exhibited in vitro antileishmanial properties in the micromolar range.
Asunto(s)
Antimaláricos/farmacología , Antiprotozoarios/farmacología , Leishmania braziliensis/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Animales , Línea Celular Tumoral/efectos de los fármacos , Eritrocitos , Humanos , Células Jurkat/efectos de los fármacos , Leishmania braziliensis/crecimiento & desarrollo , Pruebas de Sensibilidad Parasitaria , Fosfolípidos/metabolismo , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Rhodococcus sp. NCIMB 9784 accumulated 6- endo-hydroxycamphor 3 when grown on (1 R)-(+)-camphor 1 as sole carbon source. The structure of 3 has been unambiguously assigned for the first time using X-ray crystallography. A soluble cytochrome P450 hydroxylase, induced by growth on (1 R)-(+)-camphor and designated P450(camr), has been isolated from the bacterium Rhodococcus sp. NCIMB 9784. Using authentic 6- endo hydroxycamphor as standard, a cell-free system consisting of pure P450(camr) and putidaredoxin and putidaredoxin reductase from Pseudomonas putida confirmed that the enzyme hydroxylates (1 R)-(+)-camphor specifically in the 6- endoposition, in contrast to the 5- exo hydroxylation catalysed by the well-studied P450(cam) from P. putida. P450(camr) has a molecular mass of approximately 44 kDa, and a pI of 4.8.
Asunto(s)
Alcanfor/química , Alcanfor/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Rhodococcus/enzimología , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Hidroxilación , Modelos Moleculares , Rhodococcus/crecimiento & desarrollo , EstereoisomerismoRESUMEN
BACKGROUND: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. RESULTS: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. CONCLUSIONS: Erythropoietin expressed in E. coli bearing specific Asn-->Cys mutations at natural glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins.
Asunto(s)
Eritropoyetina/metabolismo , Secuencia de Aminoácidos , Eritropoyetina/síntesis química , Eritropoyetina/genética , Escherichia coli/genética , Glicosilación , Humanos , Espectrometría de Masas , Oxidación-Reducción , Mapeo Peptídico , Pliegue de Proteína , Proteínas RecombinantesRESUMEN
The enzyme 6-oxocamphor hydrolase, which catalyzes the desymmetrization of 6-oxocamphor to yield (2R,4S)-alpha-campholinic acid, has been purified with a factor of 35.7 from a wild type strain of Rhodococcus sp. NCIMB 9784 grown on (1R)-(+)-camphor as the sole carbon source. The enzyme has a subunit molecular mass of 28,488 Da by electrospray mass spectrometry and a native molecular mass of approximately 83,000 Da indicating that the active protein is trimeric. The specific activity was determined to be 357.5 units mg(-)1, and the K(m) was determined to be 0.05 mm for the natural substrate. The N-terminal amino acid sequence was obtained from the purified protein, and using this information, the gene encoding the enzyme was cloned. The translation of the gene was found to bear significant homology to the crotonase superfamily of enzymes. The gene is closely associated with an open reading frame encoding a ferredoxin reductase that may be involved in the initial step in the biodegradation of camphor. A mechanism for 6-oxocamphor hydrolase based on sequence homology and the known mechanism of the crotonase enzymes is proposed.
Asunto(s)
Alcanfor/metabolismo , Hidrolasas/genética , Hidrolasas/metabolismo , Rhodococcus/enzimología , Rhodococcus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biodegradación Ambiental , Cromatografía por Intercambio Iónico , Clonación Molecular , Enoil-CoA Hidratasa/química , Enoil-CoA Hidratasa/genética , Ferredoxina-NADP Reductasa/genética , Hidrolasas/química , Cinética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Rhodococcus/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The development of efficient, fast, flexible and general synthetic routes to glycopolymers is an ongoing challenge and much progress has been made in recent years. Chemical coupling methods have become increasingly sophisticated to fine-tune reactivity of reagents by fortuitous choices of anomeric activating group and protecting groups. As a result, oligosaccharide synthesis has become more predictable and reliable even to the extent that first examples of saccharide library syntheses in solution and on the solid phase have been published. In biology, the repertoire of biocatalysts that can be used for glycoside synthesis is ever-increasing, and enzyme-catalysed glycosylation steps have been successfully incorporated into synthetic strategies.
Asunto(s)
Biopolímeros/química , Glucosa/química , Animales , Secuencia de Carbohidratos , Glicósidos/síntesis química , Glicósidos/química , Humanos , Datos de Secuencia MolecularRESUMEN
The biochemical characterization of bacterial glycosyltransferases involved in the assembly of cell-wall-associated polysaccharides is often hindered by the lack of the appropriate undecaprenyl-pyrophosphate-linked acceptor substrate. In order to find a suitable synthetic substrate for the alpha1,3-mannosyltransferase AceA from Acetobacter xylinum, phytanyl-pyrophosphate-linked cellobiose was prepared. In the presence of GDP-[14C]mannose and recombinant AceA, the phytanyl-pyrophosphate-linked cellobiose afforded a 14C-labeled trisaccharide that was sensitive to alpha-mannosidase degradation in a fashion analogous to the natural undecaprenyl-pyrophosphate-linked cellobiose substrate. These results suggest that phytanyl-pyrophosphate-linked oligosaccharides may be useful substrates for other important bacterial glycosyltransferases.
Asunto(s)
Manosiltransferasas/metabolismo , Acetobacter/enzimología , Celobiosa/química , Celobiosa/metabolismo , Estructura Molecular , Oligosacáridos de Poliisoprenil Fosfato/química , Oligosacáridos de Poliisoprenil Fosfato/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Two structurally related beta-lactams form different covalent complexes upon reaction with porcine elastase. The high resolution x-ray structures of these two complexes provide a clear insight into the mechanism of the reaction and suggest the design of a new class of serine protease inhibitors that resist enzyme reactivation by hydrolysis of the acyl intermediate. The presence of a hydroxyethyl substituent on the beta-lactam ring provides a new reaction pathway resulting in the elimination of the hydroxyethyl group and the formation of a stabilizing conjugated double bond system. In contrast, the presence of a diethyl substituent on the beta-lactam ring leads to addition of water. The two enzyme complexes show very different binding modes in the enzyme active site.
Asunto(s)
Elastasa Pancreática/química , Inhibidores de Serina Proteinasa/química , beta-Lactamas/farmacología , Animales , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Estructura Molecular , Elastasa Pancreática/antagonistas & inhibidores , Unión Proteica , Inhibidores de Serina Proteinasa/farmacología , PorcinosRESUMEN
The preparation of the conserved core structure of asparagine-linked oligosaccharides found in eukaryotic glycoproteins is an important step towards the synthesis of homogeneous neoglycoproteins. So far, however, the convenient generation of the Manbeta4GlcNAcbeta4GlcNAc (Gn2M) core trisaccharide has proved to be a major obstacle because of the inherent difficulties associated with the synthesis of beta-mannosides. Here we report the overproduction in Escherichia coli of full-length and transmembrane-deleted yeast beta-1, 4-mannosyltransferases as novel N-terminal fusions bearing a decahistidinyl sequence and the minimal human Myc epitope. The recombinant enzymes were highly active and were amenable to immobilisation by nickel(II) chelation and to immunodetection with an anti-Myc monoclonal antibody. The immobilised, transmembrane-deleted enzyme exhibited an apparent Km of 14 microM for the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to those previously reported for native and recombinant yeast beta-1, 4-mannosyltransferases with, respectively, the natural dolichyl-linked acceptor and PPGn2. The validity of the reaction product was confirmed by chromatographic and spectroscopic analysis.
Asunto(s)
Enzimas Inmovilizadas/biosíntesis , Manosiltransferasas/biosíntesis , Oligosacáridos/biosíntesis , Secuencia de Carbohidratos , Cromatografía de Afinidad , Escherichia coli/genética , Humanos , Manosiltransferasas/química , Manosiltransferasas/genética , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Significant progress has recently been achieved in the use of glycosidases and glycosyltransferases as synthetic tools. Glycosidases have been used to synthesize trisaccharides with a reasonable overall yield, as well as high-mannose neoglycoconjugates. Studies on glycosyltransferases have defined reaction mechanisms and demonstrated reasonable substrate tolerance of these enzymes. Effective methodology for the synthesis of defined glycoproteins has also been demonstrated.
Asunto(s)
Glicoproteínas/biosíntesis , Glicósido Hidrolasas/metabolismo , Glicósidos/biosíntesis , Glicosiltransferasas/metabolismo , Oligosacáridos/biosíntesis , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicoconjugados/biosíntesis , Datos de Secuencia Molecular , Estructura Molecular , Especificidad por SustratoRESUMEN
Recombinant human erythropoietin (rhEPO) is an important glycoprotein hormone which has been successfully used in the treatment of anaemia. To facilitate the rapid evaluation of wild-type and mutant forms of rhEPO in structure-function studies, we have developed an expression system in which the recombinant hormone is tagged at the C-terminus with a c-myc peptide. One-step affinity purification of culture supernatants on an anti-myc antibody column yielded proteins which were greater than 50% pure with a specific activity of 300,000 U/mg, in agreement with the value of wild-type protein. We conclude that the additional myc-peptide does not affect receptor binding. The expression system was used to study three mutants in which the N-glycosylation sites were changed to cysteines (Asn24Cys, Asn38Cys and Asn83Cys). Specific activities of these cysteine mutants were significant, but reduced (60%, 22% and 70%, respectively), compared to wild-type. The reduction in specific activity may be due to reduced stability of the mutant proteins.
Asunto(s)
Células COS/metabolismo , Eritropoyetina/genética , Eritropoyetina/aislamiento & purificación , Expresión Génica , Mutagénesis Sitio-Dirigida , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía de Afinidad , ADN Complementario/aislamiento & purificación , Vectores Genéticos , Glicosilación , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc , Proteínas Recombinantes de Fusión/aislamiento & purificación , TransfecciónRESUMEN
Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer.
Asunto(s)
Bacteriorodopsinas/metabolismo , Proteínas de la Membrana/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Dicroismo Circular , Dimiristoilfosfatidilcolina/metabolismo , Fluorometría , Concentración de Iones de Hidrógeno , Cinética , Liposomas/metabolismo , Modelos Químicos , Éteres Fosfolípidos/metabolismo , Desnaturalización Proteica , TermodinámicaRESUMEN
The regeneration kinetics of the integral membrane protein bacteriorhodopsin have been investigated in a lipid-based refolding system. Previous studies on bacteriorhodopsin regeneration have involved detergent-based systems, and in particular mixed dimyristoylphosphatidylcholine (DMPC)/CHAPS micelles. Here, we show that the short chain lipid dihexanoylphosphatidylcholine (DHPC) can be substituted for the detergent CHAPS and that bacteriorhodopsin can be regenerated to high yield in mixed DMPC/DHPC micelles. Bacteriorhodopsin refolding kinetics are measured in the mixed DMPC/DHPC micelles. Rapid, stopped flow mixing is employed to initiate refolding of denatured bacterioopsin in SDS micelles with mixed DMPC/DHPC micelles and time-resolved fluorescence spectroscopy to follow changes in protein fluorescence during folding. Essentially identical refolding kinetics are observed for mixed DMPC/CHAPS and mixed DMPC/DHPC micelles. Only one second-order retinal/apoprotein reaction is identified, in which retinal binds to a partially folded apoprotein intermediate, and the free energy of this retinal binding reaction is found to be the same in both types of mixed micelles. Formation of the partially folded apoprotein intermediate is a rate-limiting step in protein folding and appears to be biexponential. Both apparent rate constants are found to be dependent on the relative proportion of DMPC present in the mixed DMPC/DHPC micelles as well as on the pH of the aqueous phase. Increasing the DMPC concentration should increase the bending rigidity of the amphiphilic bilayer, and this is found to slow the rate of formation of the partially folded apoprotein intermediate. Increasing the mole fraction of DMPC from 0.3 to 0.6 slows the two apparent rate constants associated with formation of this intermediate from 0.29 and 0.031 to 0.11 and 0.013 s-1, respectively. Formation of the intermediate also slows with increasing pH, from 0.11 and 0.013 s-1 at pH 6 to 0.033 and 0.0053 s-1 at pH 8. Since this pH change has no known effect on the phase behavior of lecithins, this is more likely to represent a direct effect on the protein itself. Thus, it appears to be possible to control the rate-limiting process in bacterioopsin folding through both bilayer bending rigidity and pH.
Asunto(s)
Bacteriorodopsinas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/química , Pliegue de Proteína , Ácidos Cólicos/farmacología , Dimiristoilfosfatidilcolina/farmacología , Concentración de Iones de Hidrógeno , Cinética , Membrana Dobles de Lípidos/farmacología , Liposomas/metabolismo , Modelos Químicos , Éteres Fosfolípidos/farmacología , Retinaldehído/metabolismo , Retinaldehído/farmacología , Espectrometría de Fluorescencia , Espectrofotometría , TermodinámicaRESUMEN
The chemical synthesis of the beta-mannosyl linkage of N-glycans has presented a great challenge to synthetic carbohydrate chemists. We have therefore investigated the application of beta-mannosyltransferases to the preparative synthesis N-linked core oligosaccharides. In this paper we report the chemoenzymatic synthesis of beta-D-mannopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose on a preparative scale using a phytanyl-linked acceptor in the presence of a recombinant beta-(1-->4)-mannosyltransferase.
Asunto(s)
Disacáridos/síntesis química , Manósidos/síntesis química , Manosiltransferasas/metabolismo , Trisacáridos/síntesis química , Secuencia de Carbohidratos , Enzimas Inmovilizadas/metabolismo , Escherichia coli/genética , Proteínas Fúngicas/genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Ácido Fitánico/análogos & derivados , Ácido Fitánico/química , Proteínas Recombinantes/metabolismoRESUMEN
The factors driving folding and assembly of integral membrane proteins are largely unknown. In order to determine the role that the retinal chromophore plays in assembly of bacteriorhodopsin, we have determined the kinetics and thermodynamics of retinal binding during regeneration of bacteriorhodopsin, from denatured apoprotein, in vitro. Regeneration is initiated by rapid, stopped-flow, mixing of the denatured apoprotein bacterioopsin in sodium dodecyl sulfate micelles with mixed detergent/lipid micelles containing retinal. Regeneration kinetics are measured by time-resolving changes in protein fluorescence. The dependence of each kinetic component on retinal concentration is determined. Only one experimentally observed rate constant is dependent on retinal concentration, leading to identification of only one second-order reaction involving retinal and bacterioopsin. This reaction occurs after a rate-limiting step in bacterioopsin folding, and results in formation of a noncovalent retinal/protein complex. The free energy change of this retinal binding step is determined, showing that thermodynamic information can be obtained on transient intermediates involved in membrane protein regeneration.
Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Retinaldehído/metabolismo , Halobacterium/metabolismo , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Micelas , Unión Proteica , Pliegue de Proteína , TermodinámicaRESUMEN
Protein glycosylation is a common posttranslational modification that produces glycoproteins that are highly complex in terms of both their structure and in their function. Systematic structure-function studies of such glycoproteins require synthetic methods that can produce homogeneous glycoproteins with defined oligosaccharide sidechains.
Asunto(s)
Glicoproteínas/biosíntesis , Oligosacáridos/metabolismo , Glicoproteínas/síntesis química , Glicoproteínas/metabolismo , GlicosilaciónRESUMEN
Dolichol is utilized in vivo as an unusually large anchor on which the precursor for N-linked oligosaccharides is assembled by a series of glycosyltransferases. The role of dolichol in enzyme substrate recognition is investigated. Thus the biosynthetic intermediate NN'-diacetylchitobiose was chemically linked to either dolichol or the much shorter fully saturated tetraisoprenoid phytanol. Both lipids were used as substrates by a recombinant, soluble beta-1,4-mannosyltransferase. beta-[3H]Mannosylated lipids from this reaction were then used as substrates for the subsequent mannosyltransferases from yeast or rat liver microsomes. It was found that both the dolichyl- and phytanyl-linked substrates were easily mannosylated to form Man5GlcNAc2, with some further mannosylation to Man7GlcNAc2 and Man9GlcNAc2 at low concentrations of lipid-linked substrate. It is concluded that dolichol is not necessary in vitro as part of the substrate for the mannosyltransferases in the biosynthetic pathway for N-glycosylation.