RESUMEN
PcFK1 is a member of the cysteine knot inhibitor family that displays anti-malarial properties. The naturally occurring molecule is â¼ 40 amino acids in length and forms a highly constrained 3D structure due to the presence of 3 disulfide and multiple intra-molecular H-bonds. Recent experimental studies on PcFK1 wild-type and mutants, where the cystiene residues of each disulfide bond were mutated into serine residues, suggest that alterations to these structural constraints can give rise to sizeable differences in SAR. To better understand the relationship between the dynamic inhibitor 3D structure, biophysical and biological properties we have performed solution based molecular dynamics calculations over 150 ns using the CHARMM forcefield. We have analyzed the theoretical trajectory in a systematic way using principal components analysis, which allows us to identify the correlated nature of the protein loop, turn and sheet movements. We have identified the key molecular motions that give rise to the differing SAR which has helped to more precisely direct our ongoing SAR studies in this important therapeutic area.
Asunto(s)
Antimaláricos/química , Modelos Moleculares , Conformación Molecular , Simulación de Dinámica Molecular , Venenos de Araña/química , Temperatura , Antimaláricos/farmacología , Plasmodium/efectos de los fármacos , Estabilidad Proteica , Venenos de Araña/farmacología , Relación Estructura-ActividadRESUMEN
Psalmopeotoxin I and II (PcFK1 and PcFK2), an anti-malarial peptide first extracted from Psalmopoeus cambridgei was synthesized and characterized. Both peptides belong to the Inhibitor Cystine Knot (ICK) superfamily, containing three disulfide bridges. The six cysteine residues are conserved similar to other members of the ICK superfamily, suggesting their critical role for either folding or function. In this study, the peptides were synthesized using Fmoc solid-phase peptide synthesis (SPPS). The three disulfide bonds of were constructed by regioselective and random oxidative approaches. The resulting disulfide bond patterns were verified by the HPLC-MS analysis of intact peptides and by the disulfide bond mapping using tryptic digestion. Implications of the disulfide bonds on the biophysical and biological properties of PcFKs were studied using three disulfide mutants in which a particular pair of cysteines was replaced with two isosteric serine residues. Structures and biophysical characteristics of all variants were studied using far-UV CD and fluorescence spectroscopy. Biological activities of all variants were evaluated using antiplasmodial assay against the K1 multi-drug-resistant strain of P. falciparum. The experimental results showed that the three disulfide bridges could not be correctly synthesized by the random oxidative strategy. Structural and biophysical analyses revealed that all variants had similar structures to the twisted beta-sheet. However, the studies of disulfide bond removal indicated that each disulfide bond had different effects on both biophysical and biological activities of PcFKs. Correlation of biophysical parameters and biological activities showed that both PcFKs may have different mechanisms of actions for antiplasmodial activity.
Asunto(s)
Antimaláricos/química , Antimaláricos/metabolismo , Venenos de Araña/química , Venenos de Araña/metabolismo , Arañas/química , Secuencia de Aminoácidos , Animales , Antimaláricos/uso terapéutico , Cisteína/química , Cisteína/metabolismo , Motivos Nodales de Cisteina , Disulfuros/química , Malaria/tratamiento farmacológico , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Péptidos/uso terapéutico , Conformación Proteica , Alineación de Secuencia , Venenos de Araña/genética , Venenos de Araña/uso terapéuticoRESUMEN
We studied the physical and biophysical affects of the nonionic surfactants polysorbate 20 and 80 and their mechanism of interaction using darbepoetin alfa, a 4-helix bundle protein, as the exemplary protein. Differences were observed between the abilities of the polysorbates to prevent surface loss/aggregation and correlated with each polysorbates initiation of micelle formation prior to the critical micelle concentration (CMC). The biophysical properties monitored by far-UV circular dichroism (CD) and tryptophan (Trp) fluorescence showed effects due to polysorbates, but were not correlated with their CMC. At a constant protein concentration PS-80 induced alpha-helix in the protein with a maximal effect at 15:1 molar ratio of PS-80/protein. PS-20 initially induced alpha-helix with a maximal effect at 1.5:1 ratio followed by a decrease in the alpha-helix content. PS-80 had no effect on near-UV CD but increased Trp fluorescence only at the 150:1 polysorbate/protein ratio. PS-20 decreased the near-UV CD and Trp fluorescence. Thermodynamic studies by isothermal titration calorimetry (ITC) demonstrated that the protein interacts with monomeric polysorbate, but not with polysorbate micelles. The data suggest that the polysorbates differentially interact with the protein and that the biophysical effects are dependent on the structure of the polysorbate and the polysorbate to protein ratio.
Asunto(s)
Eritropoyetina/análogos & derivados , Polisorbatos/química , Tensoactivos/química , Calorimetría , Dicroismo Circular , Darbepoetina alfa , Eritropoyetina/química , Conformación Proteica , Estabilidad Proteica , Termodinámica , Volumetría , UltracentrifugaciónRESUMEN
Perturbing the structure of the Pin1 WW domain, a 34-residue protein comprised of three beta-strands and two intervening loops has provided significant insight into the structural and energetic basis of beta-sheet folding. We will review our current perspective on how structure acquisition is influenced by the sequence, which determines local conformational propensities and mediates the hydrophobic effect, hydrogen bonding, and analogous intramolecular interactions. We have utilized both traditional site-directed mutagenesis and backbone mutagenesis approaches to alter the primary structure of this beta-sheet protein. Traditional site-directed mutagenesis experiments are excellent for altering side-chain structure, whereas amide-to-ester backbone mutagenesis experiments modify backbone-backbone hydrogen bonding capacity. The transition state structure associated with the folding of the Pin1 WW domain features a partially H-bonded, near-native reverse turn secondary structure in loop 1 that has little influence on thermodynamic stability. The thermodynamic stability of the Pin1 WW domain is largely determined by the formation of a small hydrophobic core and by the formation of desolvated backbone-backbone H-bonds enveloped by this hydrophobic core. Loop 1 engineering to the consensus five-residue beta-bulge-turn found in most WW domains or a four-residue beta-turn found in most beta-hairpins accelerates folding substantially relative to the six-residue turn found in the wild type Pin1 WW domain. Furthermore, the more efficient five- and four-residue reverse turns now contribute to the stability of the three-stranded beta-sheet. These insights have allowed the design of Pin1 WW domains that fold at rates that approach the theoretical speed limit of folding.
Asunto(s)
Pliegue de Proteína , Proteínas/química , Proteínas/metabolismo , Cinética , Modelos Moleculares , Conformación Proteica , TermodinámicaRESUMEN
In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.
Asunto(s)
Disulfuros/química , Inmunoglobulina G/química , Cadenas lambda de Inmunoglobulina/química , Cristalografía por Rayos X , Humanos , Oxidación-Reducción , Isoformas de Proteínas/química , Estructura Cuaternaria de Proteína/fisiología , Relación Estructura-ActividadRESUMEN
This study focuses on the development and application of biophysical methodology to characterize conformations of Epogen and Eprex, the injectable formulations of recombinant human Epoetin alfa produced by different manufacturers and commonly used for the treatment of renal anemia. In these studies Eprex, from prefilled syringes, and Epogen bulk product formulated in a buffer similar to the Eprex formulation, were purified by anion-exchange chromatography. Analytical ultracentrifugation studies of the purified main peak from each sample demonstrated that Epogen contains a single component with an s value of 2.51 while Eprex contains a single component with the same molecular weight but with an s value of 2.44 suggesting a slight difference in hydrodynamic structure. The degree of alpha-helicity was compared by far-UV circular dichroism and shown to contain slight differences. Intrinsic tryptophan fluorescence and near-UV circular dichroism were assessed and demonstrated additional differences between the proteins. Finally, the global stability of the proteins was monitored using thermal unfolding monitored by far-UV circular dichroism. The Epoetin alfa of Epogen demonstrated complete reversibility while the Epoetin alfa purified from Eprex demonstrated only 80%-85% thermal reversibility when heated to 100 degrees C. Together the data indicate that the proteins are not structurally identical.
Asunto(s)
Eritropoyetina/química , Algoritmos , Fenómenos Biofísicos , Biofisica , Carbohidratos/análisis , Dicroismo Circular , Composición de Medicamentos , Industria Farmacéutica , Electroforesis Capilar , Epoetina alfa , Espectroscopía de Resonancia Magnética , Peso Molecular , Polisacáridos/química , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Termodinámica , UltracentrifugaciónRESUMEN
Amide backbone and sidechain mutagenesis data can be used in combination with kinetic and thermodynamic measurements to understand the energetic contributions of backbone hydrogen bonding and the hydrophobic effect to the acquisition of beta-sheet structure. For example, it has been revealed that loop 1 of the WW domain forms in the transition state, consistent with the emerging theme that reverse turn formation is rate limiting in beta-sheet folding. A distinct subset of WW domain residues principally influences thermodynamic stability by forming hydrogen bonds and hydrophobic interactions that stabilize the native state. Energetic data and sequence mining reveal that only a small subset of the molecular information contained in sequences or observed in high-resolution structures is required to generate folded functional beta-sheets, consistent with evolutionary robustness.
Asunto(s)
Pliegue de Proteína , Estructura Secundaria de Proteína/fisiología , Termodinámica , Animales , Humanos , Cinética , Estructura Terciaria de ProteínaRESUMEN
Low molecular weight peptidomimetics with simple amphiphilic sequences can help to elucidate the structures of cross beta-sheet assemblies, such as amyloid fibrils. The peptidomimetics described herein comprise a dibenzofuran template, two peptide strands made up of alternating hydrophilic and hydrophobic residues, and carboxyl termini, each of which can be varied to probe the structural requirements for beta-sheet self-assembly processes. The dibenzofuran template positions the strands approximately 10 A apart, allowing corresponding hydrophobic side chains in the strands to pack into a collapsed U-shaped structure. This conformation is stabilized by hydrophobic interactions, not intramolecular hydrogen bonds. Intermolecular stacking of the collapsed peptidomimetics, enabled by intermolecular hydrogen bonding and hydrophobic interactions, affords 25-27 A wide protofilaments having a cross beta-sheet structure. Association of protofilaments, mediated by the dibenzofuran substructures and driven by the hydrophobic effect, affords 50-60 A wide filaments. These widths can be controlled by changing the length of the peptide strands. Further assembly of the filaments into fibrils or ribbons can be controlled by modification of the template, C-terminus, and buffer ion composition.
Asunto(s)
Péptidos/química , Proteínas/química , Amidas/química , Benzofuranos/química , Materiales Biomiméticos/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
The contribution of backbone-backbone hydrogen bonds (H-bonds) to protein folding energetics has been controversial. This is due, at least in part, to the inability to perturb backbone-backbone H-bonds by traditional methods of protein mutagenesis. Recently, however, protein backbone mutagenesis has become possible with the development of chemical and biological methods to replace individual amides in the protein backbone with esters. Here, we review the use of amide-to-ester mutation as a tool to evaluate the contribution of backbone-backbone H-bonds to protein folding kinetics and thermodynamics.
Asunto(s)
Modelos Moleculares , Pliegue de Proteína , Amidas/química , Enlace de Hidrógeno , Cinética , Mutación , TermodinámicaRESUMEN
An amide-to-ester backbone substitution in a protein is accomplished by replacing an alpha-amino acid residue with the corresponding alpha-hydroxy acid, preserving stereochemistry, and conformation of the backbone and the structure of the side chain. This substitution replaces the amide NH (a hydrogen bond donor) with an ester O (which is not a hydrogen bond donor) and the amide carbonyl (a strong hydrogen bond acceptor) with an ester carbonyl (a weaker hydrogen bond acceptor), thus perturbing folding energetics. Amide-to-ester perturbations were used to evaluate the thermodynamic contribution of each hydrogen bond in the PIN WW domain, a three-stranded beta-sheet protein. Our results reveal that removing a hydrogen bond donor destabilizes the native state more than weakening a hydrogen bond acceptor and that the degree of destabilization is strongly dependent on the location of the amide bond replaced. Hydrogen bonds near turns or at the ends of beta-strands are less influential than hydrogen bonds that are protected within a hydrophobic core. Beta-sheet destabilization caused by an amide-to-ester substitution cannot be directly related to hydrogen bond strength because of differences in the solvation and electrostatic interactions of amides and esters. We propose corrections for these differences to obtain approximate hydrogen bond strengths from destabilization energies. These corrections, however, do not alter the trends noted above, indicating that the destabilization energy of an amide-to-ester mutation is a good first-order approximation of the free energy of formation of a backbone amide hydrogen bond.
Asunto(s)
Amidas/química , Aminoácidos/química , Ésteres/química , Pliegue de Proteína , Secuencia de Aminoácidos , Dipéptidos/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Peptidilprolil Isomerasa de Interacción con NIMA , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Isomerasa de Peptidilprolil/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
[reaction: see text] The solid-phase assembly of heterocyclic amino acids enabled the total synthesis of numerous diastereoisomers of tenuecyclamides A-D, establishing or correcting the stereochemistry of each natural product. This strategy provides a very efficient route to synthesize thiazole- and oxazole-containing macrolactams from heterocyclic amino acids that are readily prepared from Fmoc-alpha-amino acids. This methodology appears to be broadly applicable to the synthesis of natural product libraries incorporating unnatural heterocyclic amino acid residues for the purpose of drug discovery.
Asunto(s)
Aminoácidos/química , Péptidos Cíclicos/síntesis química , Tiazoles/química , Tiazoles/síntesis química , Catálisis , Indicadores y Reactivos , Estructura Molecular , EstereoisomerismoRESUMEN
Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small beta-sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic cluster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a beta-sheet protein.
Asunto(s)
Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Pliegue de Proteína , Amidas/química , Amidas/metabolismo , Ésteres/química , Ésteres/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutación/genética , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
[reaction: see text] The preparation of depsi-peptides, amide-to-ester-substituted peptides used to probe the role of hydrogen bonding in protein folding energetics, is accomplished by replacing specific l-alpha-amino acid residues by their alpha-hydroxy acid counterparts in a solid-phase synthesis employing a t-Boc strategy. Herein we describe the efficient stereoselective synthesis of all 19 appropriately protected alpha-hydroxy acid equivalents of the l-alpha-amino acids, employing commercially available materials, expanding the number of available alpha-hydroxy acids from 9 to 19.
Asunto(s)
Aminoácidos/química , Hidroxiácidos/química , Péptidos/síntesis química , Catálisis , Indicadores y Reactivos , Estructura Molecular , Biosíntesis de Péptidos , EstereoisomerismoRESUMEN
The transthyretin (TTR) amyloidoses are human diseases in which the misfolded TTR protein aggregates in tissues with subsequent visceral, peripheral, and autonomic nerve dysfunction. Recent reports have stressed the importance of oligomeric intermediates as major cytotoxic species in various forms of amyloidogenesis. We have examined the cytotoxic effects of several quaternary structural states of wild-type and variant TTR proteins on cells of neural lineage. TTR amyloid fibrils and soluble aggregates >100 kDa were not toxic. Incubation of TTR under the conditions of the cell assay and analysis by size-exclusion chromatography and SDS/PAGE reveal that monomeric TTR or relatively small, rapidly formed aggregates of a maximum size of six subunits were the major cytotoxic species. Small molecules that stabilize the native tetrameric state were shown to prevent toxicity. The studies are consistent with a model in which the misfolded TTR monomer rapidly aggregates to form transient low molecular mass assemblies (<100 kDa) that are highly cytotoxic in tissue culture.
Asunto(s)
Amiloidosis/patología , Prealbúmina/química , Prealbúmina/metabolismo , Amiloidosis/genética , Línea Celular Tumoral , Supervivencia Celular , Cromatografía en Gel , Variación Genética , Humanos , Cinética , Sustancias Macromoleculares , Neuroblastoma , Prealbúmina/genética , Estructura Cuaternaria de ProteínaRESUMEN
Among the most common interaction motifs between nuclear proteins is the recognition of one or more amphipathic helices. In an effort to determine principles behind this recognition, we have investigated the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of the CREB-binding protein, CBP. The two proteins use relatively small portions of their primary sequences to form a single synergistically folded domain consisting of six intertwined alpha-helices, three from each protein. Neither of the component polypeptides forms a cooperatively folded domain in isolation. However, a considerable amount of residual secondary structure remains in the isolated CBP domain according to CD spectroscopy. Chemical denaturation, differential scanning calorimetry, and ANS binding experiments demonstrate that the isolated CBP domain is not entirely unfolded but forms a helical state with the characteristics of a molten globule. Mutations probing the functional and energetic significance of a buried intermolecular Arg-Asp salt bridge in the interface of the protein complex suggest that these residues are tuned for functional discrimination and not strictly for binding affinity or stability. These results suggest a mechanism for formation of the complex where the unfolded ACTR domain interacts with the partly folded CBP domain in a rapid and specific manner to form the final stable complex.
Asunto(s)
Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Naftalenosulfonatos de Anilina , Sitios de Unión , Proteína de Unión a CREB , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Colorantes Fluorescentes , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Coactivador 3 de Receptor Nuclear , Péptidos/química , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sensibilidad y Especificidad , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Activación Transcripcional , Ultracentrifugación , Urea/farmacologíaRESUMEN
Early-onset torsion dystonia is an autosomal dominant movement disorder that has been linked to the deletion of one of a pair of glutamic acid residues in the protein torsinA (E(302/303); DeltaE-torsinA). In transfected cells, DeltaE-torsinA exhibits similar biochemical properties to wild type (WT)-torsinA, but displays a distinct subcellular localization. Primary structural analysis of torsinA suggests that this protein is a membrane-associated member of the AAA family of ATP-binding proteins. However, to date, neither WT- nor DeltaE-torsinA has been obtained in sufficient quantity and purity to permit detailed biochemical and biophysical characterization. Here, we report a baculovirus expression system that provides milligram quantities of purified torsin proteins. Recombinant WT- and DeltaE-torsinA were found to be membrane-associated glycoproteins that required detergents for solubilization and purification. Analysis of the biophysical properties of WT- and DeltaE-torsinA indicated that both proteins were folded monomers in solution that exhibited equivalent denaturation behaviors under thermal and chaotropic (guanidinium chloride) stress. Additionally, both forms of torsinA were found to display ATPase activity with similar k(cat) and K(m) values. Collectively, these data reveal that torsinA is a membrane-associated ATPase and indicate that the DeltaE(302/303) dystonia-associated mutation in this protein does not cause gross changes in its catalytic or structural properties. These findings are consistent with a disease mechanism in which DeltaE-torsinA promotes dystonia through a gain rather than loss of function. The recombinant expression system for torsinA proteins described herein should facilitate further biochemical and structural investigations to test this hypothesis.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Distonía Muscular Deformante/metabolismo , Variación Genética , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/química , Proteínas Recombinantes/química , Adenosina Trifosfatasas/química , Animales , Baculoviridae/genética , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Dicroismo Circular , Distonía Muscular Deformante/enzimología , Distonía Muscular Deformante/genética , Vectores Genéticos , Guanidina , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Desnaturalización Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Spodoptera , Termodinámica , UltracentrifugaciónRESUMEN
Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces.
Asunto(s)
Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Oligosacáridos/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Moléculas de Adhesión Celular/metabolismo , Centrifugación por Gradiente de Densidad , Cristalización , Cristalografía por Rayos X , Dimerización , Disacáridos/química , Disacáridos/metabolismo , Epítopos , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/metabolismo , Humanos , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Lectinas/química , Lectinas/inmunología , Lectinas/metabolismo , Lectinas Tipo C/metabolismo , Ligandos , Mananos/química , Mananos/metabolismo , Manósidos/química , Manósidos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Oligosacáridos/química , Oligosacáridos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismoRESUMEN
A versatile synthesis has been developed to functionalize the 4-(2-aminoethyl)-6-dibenzofuran propionic acid residue (1a) at the 2 and 8 positions with a variety of different substructures. The unfunctionalized version of this peptidomimetic (1a) is known to facilitate beta-hairpin formation in a variety of small peptides and proteins in aqueous solution when incorporated in place of the i + 1 and i + 2 residues of a beta-turn. In this study, we append propionate substituents on 1a at the 2 and 8 positions to successfully overcome solubility problems encountered with the incorporation of 1a in place of the i + 1 and i + 2 residues of the beta-turn in loop 1 of the WW domain. The thermodynamic stability of several WW domain analogues incorporating residues 1a and 1b was compared to that of the wild-type sequence revealing comparable DeltaG(H(2)O) unfolding values at 4 degrees C ranging from 3 to 3.6 kcal/mol. WW domains incorporating residue 1b exhibit improved solubility (exceeding 100 microM) and resistance to aggregation without compromising thermodynamic stability.
Asunto(s)
Benzofuranos/química , Isomerasa de Peptidilprolil/química , Secuencia de Aminoácidos , Benzofuranos/síntesis química , Biofisica/métodos , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Peptidilprolil Isomerasa de Interacción con NIMA , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Backbone cyclization is often used in attempts to enhance protein stability, but is not always successful as it is possible to remove stabilizing or introduce destabilizing interactions in the process. Cyclization of the PIN1 WW domain, a 34-residue three-stranded beta-sheet structure, removes a favorable electrostatic interaction between its termini. Nevertheless, optimization of the linker connecting the N- and C-termini using information based on the previously determined ensemble of NMR structures leads to beta-sheets that are more stable than those derived from the linear sequence. Linkers that are too short or too long introduce strain, likely disrupting native interactions, leading to cyclic folds that are less stable than that of the linear sequence.