RESUMEN
Lipases with high kinetic stability and enzymatic efficiency in the human gastro-intestinal tract may help against exocrine pancreatic insufficiency. Here we mimic gastric conditions to study how bile salts and pH affect the stability and activity of Thermomyces lanuginosus lipase (TlL) and its stabler variant StL using spectroscopy, calorimetry and gel electrophoresis. Both enzymes resist trypsin digestion with and without bile salts. Bile salts activate native TlL and StL equally well, bind weakly to denatured TlL and StL at lower pH and precipitate native TlL and StL at pH 4. StL refolds more efficiently than TlL from gastric pH in bile salts, regaining activity when refolding from pH as low as 1.8 and above while TlL cannot go below pH 2.6. StL also unfolds 10-40 fold more slowly in the denaturant guanidinium chloride and the anionic surfactant SDS. We ascribe StL's superior performance to general alterations in its electrostatic potential which makes it more acid-resistant. These superior properties make StL a good candidate for pancreatic enzyme replacement therapy.
Asunto(s)
Ascomicetos/enzimología , Terapia de Reemplazo Enzimático , Insuficiencia Pancreática Exocrina/terapia , Lipasa/química , Proteínas Mutantes/química , Mutación/genética , Ácidos y Sales Biliares/metabolismo , Rastreo Diferencial de Calorimetría , Insuficiencia Pancreática Exocrina/enzimología , Fármacos Gastrointestinales/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lipasa/genética , Lipasa/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Pliegue de ProteínaRESUMEN
The monomeric outer membrane protein OmpA from Escherichia coli has long served as a model protein for studying the folding and membrane insertion of ß-barrel membrane proteins. Here we report that when OmpA is refolded in limiting amounts of surfactant (close to the cmc), it has a high propensity to form folded and unfolded oligomers. The oligomers exist both in a folded and (partially) unfolded form which both dissociate under denaturing conditions. Oligomerization does not require the involvement of the periplasmic domain and is not strongly affected by ionic strength. The folded dimers can be isolated and show native-like secondary structure; they are resistant to proteolytic attack and do not dissociate in high surfactant concentrations, indicating high kinetic stability once formed. Remarkably, OmpA also forms significant amounts of higher order structures when refolding in the presence of lipid vesicles. We suggest that oligomerization occurs by domain swapping favored by the high local concentration of OmpA molecules congregating on the same micelle or vesicle. In this model, the unfolded oligomer is stabilized by a small number of intermolecular ß-strand contacts and subsequently folds to a more stable state where these intermolecular contacts are consolidated in a native-like fashion by contacts between complementary ß-strands from different molecules. Our model is supported by the ability of complementary fragments to associate with each other in vitro. Oligomerization is probably avoided in the cell by the presence of cellular chaperones which maintain the protein in a monomeric state.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Escherichia coli/química , Pliegue de Proteína , Multimerización de Proteína , Concentración Osmolar , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4-6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately -12 to approximately -5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.
Asunto(s)
Ascomicetos/enzimología , Lipasa/metabolismo , Desnaturalización Proteica , Dodecil Sulfato de Sodio/metabolismo , Tensoactivos/metabolismo , Ascomicetos/química , Estabilidad de Enzimas , Glucósidos/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lipasa/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Urea/metabolismoRESUMEN
Human transforming growth factor ß induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple "beads-on-a-string" arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits.
Asunto(s)
Sustitución de Aminoácidos/genética , Proteínas de la Matriz Extracelular/química , Mutación Missense , Multimerización de Proteína , Factor de Crecimiento Transformador beta/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dispersión del Ángulo Pequeño , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Although the beta-barrel membrane protein OmpA can be produced in a biologically active form in E. coli from co-expressed fragments, the fragments have not been demonstrated to associate in vitro. We have produced 3 complementary fragment pairs of OmpA which can associate to form a folded complex according to the SDS band-shift assay. We are able to convert 25-35% of the fragment populations to non-covalent but SDS-stable complexes. The periplasmic chaperone Skp effectively prevented this association. Two separately expressed and purified overlapping fragments of OmpA can form a protease-resistant complex that undergoes the characteristic band-shift upon heating. Our work demonstrates that although membrane insertion and folding of beta-barrel membrane proteins may be a cooperative process, the fragments can associate in vitro without any additional components. However, the low yield and slow folding rates indicate that partially unfolded or destabilized beta-sheet membrane proteins can potentially engage in many non-native interactions.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Pliegue de Proteína , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Calor , Cinética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Péptido Hidrolasas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The bacterial signal recognition particle (SRP) receptor FtsY forms a complex with the SRP Ffh to target nascent polypeptide chains to the bacterial inner membrane. How FtsY interacts with lipids and associates to the membrane is unclear. Here, we show that vesicle binding leads to partial protection against proteolytic degradation and a change in secondary structure, which differs depending on whether the lipids are simple mixtures of zwitterionic and anionic lipids, mimics of Escherichia coli lipids, or lysolipids. Lipid binding alters the stability of FtsY. Thermal unfolding of FtsY in buffer shows two transitions, one occurring at approximately 60 degrees C and the other at approximately 90 degrees C. The thermal intermediate accumulating between 60 and 90 degrees C has structural features in common with the state induced by binding to E. coli lipids. E. coli lipid extract induces a single transition around 70 degrees C, anionic lipids have no effect while cooperative unfolding is completely removed in lysolipids. Thus, the lipid environment profoundly influences the dynamic properties of FtsY, leading to three different kinds of FtsY-lipid interactions with different effects on structure, proteolytic protection, and stability, and is driven both by hydrophobic and electrostatic interactions. Trypsin digestion experiments highlight the central role of the N-domain in lipid contacts, whereas the A- and G-domains appear to play a more minor part.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/química , Lípidos de la Membrana/química , Pliegue de Proteína , Receptores Citoplasmáticos y Nucleares/química , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Lípidos de la Membrana/metabolismo , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/metabolismo , Tripsina/químicaRESUMEN
Alcohols modulate the oligomerization of membrane proteins in lipid bilayers. This can occur indirectly by redistributing lateral membrane pressure in a manner which correlates with alcohol hydrophobicity. Here we investigate the direct impact of different alcohol-water mixtures on membrane protein stability and solubility, using the two detergent-solubilized alpha-helical membrane proteins DsbB and NhaA. Both proteins precipitate extensively at intermediate concentrations of alcohols, forming states with extensive (40-60%) beta-sheet structure and affinity for the fibril-specific dye thioflavin T, although atomic force microscopy images reveal layer-like and spherical deposits, possibly early stages in a fibrillation process trapped by strong hydrophobic contacts. At higher alcohol concentrations, both DsbB and NhaA are resolubilized and form non-native structures with increased (DsbB) or decreased (NhaA) helicity compared to the native state. The alternative conformational states cannot be returned to the functional native state upon dilution of alcohol. The efficiency of precipitation and the degree to which DsbB is destabilized at low alcohol concentrations show the same correlation with alcohol hydrophobicity. Thus, in addition to their effect on the membrane, alcohols perturb membrane proteins directly by solvating the hydrophobic regions of the protein. At intermediate concentrations, this perturbation exposes hydrophobic segments but does not provide sufficient solvation to avoid intermolecular association. Resolubilization requires a reduction in the relative dielectric constant below 65 in conjunction with specific properties of the individual alcohols. We conclude that alcohols provide access to a diversity of conformations for membrane proteins but are not a priori suitable for solution studies requiring reversible denaturation of monomeric proteins.
Asunto(s)
Alcoholes/química , Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Proteínas de la Membrana/química , Conformación Proteica , Intercambiadores de Sodio-Hidrógeno/química , 2-Propanol/química , Alcoholes/clasificación , Proteínas Bacterianas/ultraestructura , Benzotiazoles , Tampones (Química) , Dicroismo Circular , Proteínas de Escherichia coli/ultraestructura , Colorantes Fluorescentes/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/ultraestructura , Microscopía de Fuerza Atómica , Modelos Químicos , Propanoles/química , Desnaturalización Proteica , Estructura Secundaria de Proteína , Intercambiadores de Sodio-Hidrógeno/ultraestructura , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Tiazoles/metabolismo , Trifluoroetanol/química , Agua/químicaRESUMEN
The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 degrees C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 degrees C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.
Asunto(s)
Proteínas de la Membrana/química , Micelas , Adhesinas de Escherichia coli/química , Fenómenos Bioquímicos , Bioquímica , Dicroismo Circular , Detergentes/química , Glucósidos/química , Calor , Iones , Cinética , Polietilenglicoles/química , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Dodecil Sulfato de Sodio/química , Solventes , Espectrometría de Fluorescencia , Temperatura , Termodinámica , Factores de Tiempo , Rayos UltravioletaRESUMEN
Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone. Their exterior is hydrophilic and their cavity is hydrophobic, which gives cyclodextrins the ability to accommodate hydrophobic molecules/moieties in the cavity. This special molecular arrangement accounts for the variety of beneficial effects cyclodextrins have on proteins, which is widely used in pharmacological applications. We have studied the interaction between beta-cyclodextrin and four non-carbohydrate-binding model proteins: ubiquitin, chymotrypsin inhibitor 2 (CI2), S6 and insulin SerB9Asp by NMR spectroscopy at varying structural detail. We demonstrate that the interaction of beta-cyclodextrin and our model proteins takes place at specific sites on the protein surface, and that solvent accessibility of those sites is a necessary but not compelling condition for the occurrence of an interaction. If this behaviour can be generalized, it might explain the wide range of different effects of cyclodextrins on different proteins: aggregation suppression (if residues responsible for aggregation are highly solvent accessible), protection against degradation (if point of attack of a protease is sterically 'masked' by cyclodextrin), alteration of function (if residues involved in function are 'masked' by cyclodextrin). The exact effect of cyclodextrins on a given protein will always be related to the particular structure of this protein.
Asunto(s)
Ciclodextrinas/química , Ciclodextrinas/metabolismo , Interpretación Estadística de Datos , Humanos , Insulina/genética , Insulina/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Péptidos/metabolismo , Proteínas de Plantas , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Quinasas S6 Ribosómicas/metabolismo , Ubiquitina/metabolismoRESUMEN
To examine the influence of contact order and stability on the refolding rate constant for two-state proteins, we have analysed the folding kinetics of the small beta-alpha-beta protein S6 and two of its circular permutants with relative contact orders of 0.19, 0.15 and 0.12. Data reveal a small but significant increase of the refolding rate constant (log k(f)) with decreasing contact order. At the same time, the decreased contact order is correlated to losses in global stability and alterations of the folding nucleus. When the differences in stability are accounted for by addition of Na2SO4 or by comparison of the folding kinetics at the transition mid-point, the dependence between log k(f) and contact order becomes stronger and follows the general correlation for two-state proteins. The observation emphasizes the combined action of topology and stability in controlling the rate constant of protein folding.
Asunto(s)
Ingeniería de Proteínas , Pliegue de Proteína , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Sitios de Unión , Ciclización , Cinética , Modelos Moleculares , Mutación/genética , Proteínas/química , Proteínas/metabolismo , Proteína S6 Ribosómica , Proteínas Ribosómicas/genética , TermodinámicaRESUMEN
Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be pH-denatured. Further, some proteins do not refold reversibly from low pH where they get trapped as aggregation prone intermediates. Here, we present an alternative approach for direct measurement of refolding rates in water, which does not rely on extrapolation. The protein is denatured in SDS, and is then mixed with alpha-cyclodextrin, which rapidly strips SDS molecules from the protein, leaving the naked unfolded protein to refold.
Asunto(s)
Pliegue de Proteína , Proteínas/química , Proteínas/metabolismo , Agua/metabolismo , alfa-Ciclodextrinas , Animales , Pollos , Ciclodextrinas/metabolismo , Ciclodextrinas/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Concentración de Iones de Hidrógeno , Isomerismo , Cinética , Muramidasa/química , Muramidasa/metabolismo , Prolina/química , Prolina/metabolismo , Desnaturalización Proteica/efectos de los fármacos , Proteína S6 Ribosómica , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Dodecil Sulfato de Sodio/metabolismo , Dodecil Sulfato de Sodio/farmacología , Termodinámica , Agua/químicaRESUMEN
For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide and propylene oxide (Breox) and dextran T-500. Between 25 and 50 degrees C, the partition coefficients of CI2 in Breox-dextran T-500 systems remain constant at neutral pH. However, there is a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 50 degrees C, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60 degrees C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the folded and the unfolded states of the protein, where the unfolded CI2 with a more hydrophobic surface is partitioned to the relatively hydrophobic Breox phase in both systems. A separation process is presented based on the partitioning behavior of unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The target protein can be recovered through (i) selective separation in Breox-dextran systems, (ii) refolding in Breox phase, and (iii) thermoseparation of primary Breox phase.
Asunto(s)
Quimotripsina/antagonistas & inhibidores , Modelos Químicos , Péptidos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Péptidos/química , Proteínas de Plantas , Polímeros , Pliegue de Proteína , TemperaturaRESUMEN
Limited solubility and precipitation of amyloidogenic sequences such as the Alzheimer peptide (beta-AP) are major obstacles to a molecular understanding of protein fibrillation and deposition processes. Here we have circumvented the solubility problem by stepwise engineering a beta-AP homology into a soluble scaffold, the monomeric protein S6. The S6 construct with the highest beta-AP homology crystallizes as a tetramer that is linked by the beta-AP residues forming intermolecular antiparallel beta-sheets. This construct also shows increased coil aggregation during refolding, and a 14-mer peptide encompassing the engineered sequence forms fibrils. Mutational analysis shows that intermolecular association is linked to the overall hydrophobicity of the sticky sequence and implies the existence of "structural gatekeepers" in the wild-type protein, that is, charged side chains that prevent aggregation by interrupting contiguous stretches of hydrophobic residues in the primary sequence.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Oligopéptidos/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Péptidos beta-Amiloides/ultraestructura , Cristalografía por Rayos X , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , SolubilidadRESUMEN
The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally stabilized by 0.1 mM Zn(2+) compared with Zn(2+)-reduced conditions achieved using 1 mM EDTA or specific replacements that affect Zn(2+) coordination. The higher stability of SEE was only partly caused by the T-cell receptor (TCR) binding regions, whereas regions in the vicinity of the major histocompatibility complex class II binding sites affected the stability to a greater extent. SEE exhibited a biphasic denaturation between pH 5.0-6.5, influenced by residues in the TCR binding regions. Interestingly, enzyme-linked immunosorbent assay, isoelectric focusing, and circular dichroism analysis indicated that conformational changes had occurred in the SEA/E chimerical constructs relative to SEA and SEE. Thus, it is proposed that the Zn(2+) binding site is very important for the stability and potency of SEA and SEE, whereas residues in the TCR binding site have a substantial influence on the molecular conformation to control specificity and function.
Asunto(s)
Enterotoxinas/química , Secuencia de Aminoácidos , Animales , Células Cultivadas , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Guanidina/farmacología , Antígenos de Histocompatibilidad Clase II/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de Aminoácido , Bazo/metabolismo , Relación Estructura-Actividad , Superantígenos/metabolismo , Linfocitos T/metabolismo , Temperatura , Termodinámica , Zinc/metabolismoRESUMEN
In several cases, inorganic salts have been used to induce partly structured states in protein folding. But what is the nature of these states: Do they represent key stepping stones in the folding process, or are they circumstantial pitfalls in the energy landscape? Here we report that, in the case of the two-state protein S6, the salt-induced collapsed state is off the usual folding routes in the sense that it is prematurely collapsed and slows down folding by several orders of magnitude. Although this species is over-compact, it is not a dead-end trap but may fold by alternative channels to the native state.
RESUMEN
Cellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Cel45 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Cel45 unfolds in GdmCl according to a simple two-state model under equilibrium conditions, it accumulates a transient intermediate during refolding. The four disulfide bonds do not contribute detectably to the stability of the native state. Cel45 is unfolded by very low concentrations of C12-LAS (1-4 mM). An analysis of 16 mutants of Cel45 shows a very weak correlation between unfolding rates in denaturant and detergent; mutants that have the same unfolding rate in GdmCl (within a factor of 1.5) vary 1,000-fold in their unfolding rates in C12-LAS. The data support a simple model for unfolding by detergent, in which the introduction of positive charges or removal of negative charges greatly increases detergent sensitivity, while interactions with the hydrophobic detergent tail contribute to a smaller extent. This implies that different detergent-mediated unfolding pathways exist, whose accessibilities depend on individual residues. Double-mutant cycles reveal that mutations in two proximal residues lead to repulsion and a destabilization greater than the sum of the individual mutations as measured by GdmCl denaturation, but they also reduce the affinity for LAS and therefore actually stabilize the protein relative to wild-type. Ligands that interact strongly with the denatured state may therefore alter the unfolding process.
Asunto(s)
Ácidos Alcanesulfónicos/química , Celulasa/química , Celulasa/metabolismo , Hongos Mitospóricos/enzimología , Tensoactivos/química , Equilibrio Ácido-Base , Guanidina/química , Cinética , Modelos Químicos , Desnaturalización Proteica , Ingeniería de Proteínas , Pliegue de ProteínaRESUMEN
The interpretation of folding rates is often rationalized within the context of transition state theory. This means that the reaction rate is linked to an activation barrier, the height of which is determined by the free energy difference between a ground state (the starting point) and an apparent transition state. Changes in the folding kinetics are thus caused by effects on either the ground state, the transition state, or both. However, structural changes of the transition state are rarely discussed in connection with experimental data, and kinetic anomalies are commonly ascribed to ground state effects alone, e.g., depletion or accumulation of structural intermediates upon addition of denaturant. In this study, we present kinetic data which are best described by transition state changes. We also show that ground state effects and transition state effects are in general difficult to distinguish kinetically. The analysis is based on the structurally homologous proteins U1A and S6. Both proteins display two-state behavior, but there is a marked difference in their kinetics. S6 exhibits a classical V-shaped chevron plot (log observed rate constant vs denaturant concentration), whereas U1A's chevron plot is symmetrically curved, like an inverted bell curve. However, S6 is readily mutated to display U1A-like kinetics. The seemingly drastic effects of these mutations are readily ascribed to transition state movements where large kinetic differences result from relatively small alterations of a common free energy profile and broad activation barriers.
Asunto(s)
Pliegue de Proteína , Proteínas de Unión al ARN , Ribonucleoproteína Nuclear Pequeña U1/química , Proteínas Ribosómicas/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Desnaturalización Proteica , Estructura Secundaria de Proteína , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Proteína S6 Ribosómica , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Termodinámica , Thermus thermophilusRESUMEN
Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information.
Asunto(s)
Mutagénesis Sitio-Dirigida , Péptidos/química , Conformación Proteica , Subtilisinas/química , Cinética , Proteínas de Plantas , Unión Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
The 64-residue chymotrypsin inhibitor 2 (CI2) folds by a two-state nucleation-condensation mechanism, whereby secondary and tertiary structure coalesce concomitantly in the transition state around Ala 16 in the helical N-cap. Permutation of the SH3-domain of alpha-spectrin apparently shifts its folding nucleus to another region of the protein, suggesting that a protein's transition state may be altered by altering the protein's connectivity. We have characterized the structure of the transition state of a circular and a permuted version of CI2 by a protein engineering study encompassing 11 mutations. Circular CI2 was obtained by the introduction of cysteines at residues 3 and 63 and linking them by disulfide bond formation. Subsequent cyanogen-bromide cleavage of the scissile bond, Met 40-Glu 41, yielded permuted CI2. Circular and permuted CI2 also fold according to a two-state mechanism. Permutation does not affect the folding rate constant, but circularization increases it 7-fold. The transition states of circular and permuted CI2 are essentially unchanged from that of wild-type CI2. Importantly, the folding nucleus around Ala16 is retained. These results complement a previous observation that the transition state for association of two CI2 fragments (residues 1-40 and 41-64, generated by CNBr cleavage) is very similar to the folding transition state of intact CI2. The similarity of rate constants for folding of wild-type and permuted CI2, and their value relative to that for the association of fragments, allows us to estimate the gain in entropy of activation on having the separate fragments linked: 18.3 cal M-1 K-1; i.e. an effective molarity of 10(4) M. The contrast between the retention of the folding nucleus on permutation of CI2 and its change for the SH3-domain of alpha-spectrin probably arises because the latter was cleaved in its folding nucleus whereas cleavage at sites other than 40-41 in CI2 is very destabilizing. Whether or not a folding nucleus can be changed probably depends on the specific protein and its permissivity to permutation.
Asunto(s)
Quimotripsina/antagonistas & inhibidores , Péptidos/química , Péptidos/genética , Pliegue de Proteína , Disulfuros/química , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Proteínas de Plantas , Desnaturalización Proteica , Ingeniería de Proteínas , Estructura Secundaria de ProteínaRESUMEN
Two-dimensional NMR spectroscopy has been used to monitor hydrogen-deuterium exchange in chymotrypsin inhibitor 2. Application of two independent tests has shown that at pH 5.3 to 6.8 and 33 to 37 degrees C, exchange occurs via an EX2 limit. Comparison of the exchange rates of a number of mutants of CI2 with those of wild-type identifies the pathway of exchange, whether by local breathing, global unfolding or a mixture of the two pathways. For a large number of residues, the exchange rates were unaffected by mutations which destabilized the protein by up to 1.9 kcal mol(-1), indicating that exchange is occurring through local fluctuations of the native state. A small number of residues were found for which the mutations had the same effect on the rate constants for exchange as on the equilibrium constant for unfolding, indicating that these residues exchange by global unfolding. These are residues that have the slowest exchange rates in the wild-type protein. We see no correspondence between these residues and residues involved in the nucleation site for the folding reaction identified by protein engineering studies. Rather, the exchange behaviour of CI2 is determined by the native structure: the most protected amide protons are located in regions of hydrogen bonding, specifically the C terminus of the alpha-helix and the centre of the beta-sheet. A number of the most slowly exchanging residues are in the hydrophobic core of the protein.