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Conformational flexibility of a model protein upon immobilization on self-assembled monolayers
Biotechnology and Bioengineering
Abstract
The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD(+), some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed.
... rotein structure with the essential mechanism for carrying enzymatic reactions. The effect of conformational flexibility on the enzyme activity was initially justified by the observation that enzyme immobilized on a carrier via a suitable spacer often resulted in better retention of activity than the enzyme immobilized without a spacer163164165166. Bigdeli et al. (2008) reported the retention of conformational flexibility of glutamate dehydrogenase, used as a model allosteric protein, upon its immobilization on self-assembled monolayers-modified gold [167]. Activity of immobilized enzyme is often influenced by binding mode. The effect of binding mode may be reflected by the number of bonds formed betwe ...
... The effect of conformational flexibility on the enzyme activity was initially justified by the observation that enzyme immobilized on a carrier via a suitable spacer often resulted in better retention of activity than the enzyme immobilized without a spacer163164165166. Bigdeli et al. (2008) reported the retention of conformational flexibility of glutamate dehydrogenase, used as a model allosteric protein, upon its immobilization on self-assembled monolayers-modified gold [167]. Activity of immobilized enzyme is often influenced by binding mode. ...
Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.
... It is a common belief that conformational flexibility provides a protein structure with the essential mechanism for carrying enzymatic reactions. The effect of conformational flexibility on the enzyme activity was initially justified by the observation that enzyme immobilized on a carrier via a suitable spacer often resulted in better retention of activity than the enzyme immobilized without a spacer [163][164][165][166]. Bigdeli et al. (2008) reported the retention of conformational flexibility of glutamate dehydrogenase, used as a model allosteric protein, upon its immobilization on self-assembled monolayers-modified gold [167]. ...
... It is a common belief that conformational flexibility provides a protein structure with the essential mechanism for carrying enzymatic reactions. The effect of conformational flexibility on the enzyme activity was initially justified by the observation that enzyme immobilized on a carrier via a suitable spacer often resulted in better retention of activity than the enzyme immobilized without a spacer [163][164][165][166]. Bigdeli et al. (2008) reported the retention of conformational flexibility of glutamate dehydrogenase, used as a model allosteric protein, upon its immobilization on self-assembled monolayers-modified gold [167]. ...
The anti-tumor effect of aconitine in melanoma cell line B16 has been studied in this paper. We found that B16 cells showed significantly reduced growth rates and increased apoptotic effects in the presence of aconitine. Furthermore, aconitine inhibited the PI3K/AKT and MAPK/ERK1/2 signaling pathways, thus regulating the levels of protein and mRNA of PCNA and apoptotic related signaling molecules. Above all, we found that aconitine showed an anti-melanoma effect in suppressing tumor growth in vivo. In conclusion, we show that aconitine may be a useful anticancer drug in the future.
... Asuri et al. demonstrated that enzymes covalently immobilized to singlewalled carbon nanotubes retained their native structures (Asuri et al. 2007). Bigdeli et al. studied the conformational flexibility of glutamate dehydrogenase when immobilized by adsorption or by covalent binding (Bigdeli et al. 2008). They proved that the enzyme retained a certain mobility while being immobilized on self-assembled monolayers. ...
Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N
α-benzoyl-l-arginine-4-nitroanilide hydrochloride (l-BAPNA), and in the presence or absence of d-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of d-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with d-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus l-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.
The development of the receptor layer of the biosensor for detecting explosive compounds is described. The covalent modification has been chosen for immobilizing E. coli nitroreductase on the gate oxide of the ion-sensitive field effect transistor (ISFET) that is comprised of silicon dioxide. The self-assembled monolayer technique has been used for immobilization. This method assumes the usage of different silanes and spacer molecules for activating the surface of SiO2. Two different immobilization strategies have been compared, one using asymmetric spacers (3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) and 4-(4-maleimidophenyl)butyric acid N-hydroxysuccinimide ester (SMPB)) and another using a symmetric glutaric dialdehyde linker both accompanied by appropriate silanes. For the first method, the dependence of functionalization efficiency on silane concentration has been studied. The sufficient density of enzyme molecules on the surface of SiO2 has been achieved at a concentration of silane of 0.0015%. The type of asymmetric linker has no influence on immobilization efficiency. The method implying glutaric dialdehyde results in higher activity of the immobilized enzyme. For this method, the immobilization procedure has been optimized. The method has been adapted for immobilization of E. coli nitroreductase inside the channel of a microfluidic system on the surface of ISFET. For this purpose, (3-aminopropyl)triethoxysilane (APTES) has been changed to the corresponding silatrane, and the concentration of the enzyme has been increased to 30 μg/mL. The optimized procedure has been successfully used to develop a biosensor for detecting explosives.
Glutamate dehydrogenase (GDH) is a well known homohexameric enzyme and its performance in immobilized form was systematically investigated in this work, in order to provide a better understanding of the multimeric enzyme immobilization effects in relation to the monomeric ones. For this purpose, GDH was immobilized on four different magnetic supports and the outcome from such immobilization was characterized in terms of their stabilization and activity. Immobilization procedures involving amine coupling via glutaraldehyde cross-linking yielded the least stable ones, even lower than free GDH, showing no recoverability. However, the immobilization procedures using larger aldehyde cross-linkers presented higher thermal stabilities than free GDH and could be recycled at least 10 times, with a nearly constant activity. Such differences in stability and activities were thoroughly evaluated in terms of the enzymatic structure, which has guided the reasoning behind the intriguing allosteric behavior of the immobilized GDH. The atypical allosteric response exhibited by MagNP-APTS/GDH, MagNP@SiO2-APTS/GDH and MagNP-APTS/Glyoxyl-Agarose/GDH led us to invoke the intrinsic disorder theory to explain the results. This theory has proved an excellent tool to guide researches on immobilized enzymes and understand its effects.
A novel ion-selective field effect transistor (ISFET)-based biosensor was developed for the detection of explosives. Escherichia coli nitroreductase (NTR) was used as the recognizing element of the biosensor to afford high specificity and sensitivity toward nitroaromatic compounds. The ISFET-based device demonstrated an analytical response toward the NTR-catalyzed reduction of several nitroaromatic substances, conjugated to the oxidation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+. Catalytic properties of nitroreductase were characterized by spectrophotometric analysis. Nitroreductase showed maximum activity toward tetryl and trinitrotoluene, and the Michaelis constants were determined to be 70 and 91 μM, respectively. The enzyme was covalently attached to the surface of ISFET using a self-assembly method. Two SiO2-surface immobilization strategies using an asymmetric spacer, 3-maleimidobenzoic acid N-hydroxysuccinimide ester, and a symmetric spacer, glutaric dialdehyde, were compared. The use of the symmetric spacer resulted in higher enzymatic activity of the immobilized nitroreductase. An NTR-functionalized complementary metal–oxide-semiconductor (CMOS)-compatible ISFET chip was combined with a microfluidic system for analyte delivery. The response of the system to several nitroaromatic compounds was examined. The device demonstrated a low detection limit and may be applied for the rapid detection of explosives in various samples.
Protein aggregates, whether amorphous or structured (amyloid), have attracted much attention in recent years and despite extensive efforts, the mechanism of their formation is poorly understood. While “natural” aggregation (polymerization) of monomers could improve the biological function of some proteins, it is usually the darker side of this phenomenon which is discussed in many studies: deleterious aggregation that could lead to loss of biological activity under in vitro conditions or cause misfolding diseases. In this review, protein aggregation has been overviewed, starting from some general concepts involved in its formation, followed by mentioning studies aimed at elucidation of its kinetics and mechanism, or characterization of intermediates that would be aggregation-prone, and finally, reporting some of the studies related to the design of methods that would control the process. Similarly, amyloid aggregates have been defined, and current methods used in their characterization have been briefly described, with an emphasis on in silico studies. Finally, identification and design of such molecules which may be effective in control of this process is discussed.
We present a new analytical approach to the strong electrostatic coupling regime (SC) that can be achieved equivalently at low temperatures, high charges, low dielectric permittivity, etc. Two geometries are analyzed in detail: one charged wall first, and then two parallel walls at small distances that can be likely or oppositely charged. In all cases, only one type of mobile counterions is present, and ensures electroneutrality (salt-free case). The method is based on a systematic expansion around the ground state formed by the two-dimensional Wigner crystal(s) of counterions at the plate(s). The leading SC order stems from a single-particle theory, and coincides with the virial SC approach that has been much studied in the last 10 years. The first correction has the functional form of the virial SC prediction, but the prefactor is different. The present theory is free of divergences and the obtained results, both for symmetrically and asymmetrically charged plates, are in excellent agreement with available data of Monte Carlo simulations under strong and intermediate Coulombic couplings. All results obtained represent relevant improvements over the virial SC estimates. The present SC theory starting from the Wigner crystal and therefore coined Wigner SC, sheds light on anomalous phenomena like the counterion mediated like-charge attraction, and the opposite-charge repulsion.
Within a mean-field treatment of the interaction between two oppositely charged plates in a salt-free solution, the distance at which a transition from an attractive to a repulsive regime appears can be computed analytically. The mean-field description, however, breaks down under strong Coulombic couplings, which can be achieved at room temperature with multivalent counterions and highly charged surfaces. Making use of the contact theorem and simple physical arguments, we propose explicit expressions for the equation of state in several situations at short distances. The possibility of Bjerrum pair formation is addressed and is shown to have profound consequences on the interactions. To complete the picture, we consider the large-distance limit, from which schematic phase diagram discriminating attractive from repulsive regions can be proposed.
Stopped flow studies of the oxidative deamination of l-glutamate by TPN and beef liver glutamate dehydrogenase were performed at pH 6.5 and 7.6. At each pH value, the initial burst slopes obey an equation of the form: e/v = Φ0 + Φ1/(TPN) + Φ2/(l-glutamate) + Φ12/(TPN)-(l-glutamate). The agreement of dissociation constants for enzyme-TPN and enzyme-l-glutamate binary complexes obtained from the Φ values with those obtained independently in equilibrium studies supports mechanisms which include random and rapid binding of substrate and coenzyme at the active site. The lack of effect of preincubation with either reactant on the transient state kinetics eliminates the possibility of any kinetically important slow isomerization steps in the formation of binary complexes. Comparison of the limiting Michaelis constants with the dissociation constants for TPN and l-glutamate shows that there is a cooperativity between coenzyme and substrate in the formation of an enzyme-TPN-l-glutamate ternary complex.
A monolayer of poly(L-lysine) (PL) is attached covalently via amide bonds to an alkanethiol self-assembled monolayer (SAM) on a gold surface. The amide bond is formed in two steps: the terminal carboxylic acid groups of an 11-mercaptoundecanoic acid (MUA) SAM are first activated to the N-hydroxysulfosuccinimide (NHSS) ester, followed by reaction of this MUA-NHSS ester monolayer with the amino groups of PL to create multiple amide bond linkages to the surface. The reactivity and packing density of the MUA-NHSS esters are investigated in detail by reacting these intermediates with ammonia (NH3). In the NH3 experiments, approximately 50% of the carboxylic acids in the MUA monolayer are converted to amides during the first cycle of this two-step surface reaction. This reaction yield of 50% is limited by the steric packing of the NHSS ester intermediate. However, after three cycles of MUA activation to the NHSS ester and reaction with NH3, nearly all of the MUA molecules (similar to 80%) are converted to amides. Polarization-modulation Fourier transform infrared reflection-absorption spectroscopy (PM-FT-IRRAS) is employed to study both the NH3 and PL reactions on the gold surface. The PM-FT-IRRAS spectrum of a covalently attached PL monolayer indicates that amide bonds are formed with the underlying MUA molecules. This conclusion is confirmed by the fact that the covalent PL monolayer resists desorption despite immersion into solutions of pH < 2 or pH > 12, Finally, the PL is derivatized with a bifunctional NHSS ester-maleimide molecule either by reaction in solution prior to covalent attachment or by reaction with PL already adsorbed to the surface. Up to 50% of the total number of lysine amino groups are converted to maleimide groups, which can be used for the subsequent attachment of sulfhydryl-containing biomolecules.
In this study, we present conformational energies for a molecular mechanical model (Parm99) developed for organic and biological molecules using the restrained electrostatic potential (RESP) approach to derive the partial charges. This approach uses the simple "generic" force field model (Parm94), and attempts to add a minimal number of extra Fourier components to the torsional energies, but doing so only when there is a physical justification. The results are quite encouraging, not only for the 34-molecule set that has been studied by both the highest level ab initio model (GVB/LMP2) and experiment, but also for the 55-molecule set for which high-quality experimental data are available. Considering the 55 molecules studied by all the force field models for which there are experimental data, the average absolute errors (AAEs) are 0.28 (this model), 0.52 (MM3), 0.57 (CHARMm [MSI]), and 0.43 kcal/mol (MMFF). For the 34-molecule set, the AAEs of this model versus experiment and nb initio are 0.28 and 0.27 kcal/mol, respectively. This is a lower error than found with MM3 and CHARMm, and is comparable to that found with MMFF (0.31 and 0.22 kcal/mol). We also present two examples of how well the torsional parameters are transferred from the training set to the test set. The absolute errors of molecules in the test set are only slightly larger than in the training set (differences of <0.1 kcal/mol). Therefore, it can be concluded that a simple "generic" force field with a limited number of specific torsional parameters can describe intra- and intermolecular interactions, although all comparison molecules were selected from our 82-compound training set. We also show how this effective two-body model can be extended for use with a nonadditive force field (NAFF), both with and without lone pairs. Without changing the torsional parameters, the use of more accurate charges and polarization leads to an increase in average absolute error compared with experiment, but adjustment of the parameters restores the level of agreement found with the additive model. After reoptimizing the psi, Phi torsional parameters in peptides using alanine dipeptide (6 conformational pairs) and alanine tetrapeptide (11 conformational pairs), the new model gives better energies than the Cornell et al. (J Am Chem Soc 1995, 117, 5179-5197) force field. The average absolute error of this model for high-level nb initio calculation is 0.82 kcal/mol for alanine dipeptide and tetrapeptide as compared with 1.80 kcal/mol for the Cornell et al. model. For nucleosides, the new model also gives improved energies compared with the Cornell et al. model. To optimize force field parameters, we developed a program called parmscan, which can iteratively scan the torsional parameters in a systematic manner and finally obtain the best torsional potentials. Besides the organic molecules in our test set, parmscan was also successful in optimizing the Psi, Phi torsional parameters in peptides to significantly improve agreement between molecular mechanical and high-level nb initio energies. (C) 2000 John Wiley & Sons, Inc.
Aspartate transcarbamoylase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allostery. This enzyme shows cooperativity between the catalytic sites, and its activity is feedback inhibited by CTP and activated by ATP. These regulatory processes involve several interfaces between catalytic and regulatory chains as well as between domains within these two types of chains. As far as the regulatory chain is concerned, its two domains are in contact through a hydrophobic interface, in which a tyrosine residue is inserted in a pocket involving two leucine residues of the allosteric domain and a valine and a leucine residue of the zinc domain. To probe the possible implication of this hydrophobic core in the CTP and ATP regulatory effect, the tyrosine was replaced by a phenylalanine through oligonucleotide-directed mutagenesis. Interestingly, the resulting mutant shows a complete inversion of the ATP effect; it is now inhibited by ATP instead of being activated by this nucleotide triphosphate. This mutant remains normally sensitive to the feedback inhibitor CTP. This result shows that the hydrophobic interface between the two domains of the regulatory chain plays an important role in the discrimination between the regulatory signals promoted by the two allosteric effectors.
Glutamate dehydrogenase in disrupted mitochondrial preparations is activated by L-leucine to a much greater extent than is the purified enzyme. A factor, or factors, responsible for modulating the sensitivity of L-leucine is lost during the purification of the enzyme. Although both cardiolipin and phosphatidylserine are inhibitors of the enzyme, only the inhibition by the former phospholipid is reversed by L-leucine. The inhibition of glutamate dehydrogenase by its binding to cardiolipin in the disrupted mitochondrial preparations and its relief by L-leucine could account for the greater sensitivity of such preparations to activation by that amino acid.
Stopped flow studies of the oxidative deamination of l-glutamate by TPN and beef liver glutamate dehydrogenase were performed at pH 6.5 and 7.6. At each pH value, the initial burst
slopes obey an equation of the form: e/v = Φ0 + Φ1/(TPN) + Φ2/(l-glutamate) + Φ12/(TPN)-(l-glutamate). The agreement of dissociation constants for enzyme-TPN and enzyme-l-glutamate binary complexes obtained from the Φ values with those obtained independently in equilibrium studies supports mechanisms
which include random and rapid binding of substrate and coenzyme at the active site. The lack of effect of preincubation with
either reactant on the transient state kinetics eliminates the possibility of any kinetically important slow isomerization
steps in the formation of binary complexes. Comparison of the limiting Michaelis constants with the dissociation constants
for TPN and l-glutamate shows that there is a cooperativity between coenzyme and substrate in the formation of an enzyme-TPN-l-glutamate ternary complex.
The influences on ion energy of membrane thickness, ion-pair formation, ``pores'' and ``carriers'' have been estimated. Only ``pores'' and ``carriers'' lower the energy barrier significantly.
The activity of bovine liver glutamate dehydrogenase is affected in several ways depending on substrate concentrations and pH. At ph 6.5 and below, both oxidative deamination and reductive amination reactions are inhibited by ADP. At pH 7.0 and above both activatory and inhibitory effects can be observed depending on substrate concentrations. The effects are explicable in terms of a model with ADP binding at both a regulatory site and competing with coenzyme at the active site. The activatory effects of ADP result from destabilization of various abortive complexes by ADP binding to its regulatory site. The concerted effects of pH and ADP lead to a potentiation of either activation effects or inhibition effects depending on conditions. A consideration of in vivo concentrations of the various substrates involved and intramitochondrial pH and adenine nucleotide levels suggests that in vivo the reductive amination reaction is favored. It is suggested that glutamate dehydrogenase may be intimately involved with regulation of the urea cycle by responding to changes in the mitochondrial ammonia levels.
To explore how distal mutations affect binding sites and how binding sites in proteins communicate, an ensemble-based model of the native state was used to define the energetic connectivities between the different structural elements of Escherichia coli dihydrofolate reductase. Analysis of this model protein has allowed us to identify two important aspects of intramolecular communication. First, within a protein, pair-wise couplings exist that define the magnitude and extent to which mutational effects propagate from the point of origin. These pair-wise couplings can be identified from a quantity we define as the residue-specific connectivity. Second, in addition to the pair-wise energetic coupling between residues, there exists functional connectivity, which identifies energetic coupling between entire functional elements (i.e., binding sites) and the rest of the protein. Analysis of the energetic couplings provides access to the thermodynamic domain structure in dihydrofolate reductase as well as the susceptibility of the different regions of the protein to both small-scale (e.g., point mutations) and large-scale perturbations (e. g., binding ligand). The results point toward a view of allosterism and signal transduction wherein perturbations do not necessarily propagate through structure via a series of conformational distortions that extend from one active site to another. Instead, the observed behavior is a manifestation of the distribution of states in the ensemble and how the distribution is affected by the perturbation.
The problem of the propagation of conformational changes over long distances or through a closely packed protein is shown to fit a model of a ligand-induced conformational change between two protein states selected by evolution. Moreover, the kinetics of the pathway between these states is also selected so that the energy of ligand binding and the speed of the transition between conformational states are physiologically appropriate. The crystallographic data of a wild-type aspartate receptor that has negative cooperativity and a mutant that has no cooperativity but has native transmembrane signaling are shown to support this model.
Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Unlike GDH from bacteria, mammalian GDH exhibits negative cooperativity with respect to coenzyme, activation by ADP, and inhibition by GTP. Presented here are the structures of apo bovine GDH, bovine GDH complexed with ADP, and the R463A mutant form of human GDH (huGDH) that is insensitive to ADP activation. In the absence of active site ligands, the catalytic cleft is in the open conformation, and the hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals. This is consistent with the fact that ADP promotes aggregation in solution. ADP is shown to bind to the second, inhibitory, NADH site yet causes activation. The beta-phosphates of the bound ADP interact with R459 (R463 in huGDH) on the pivot helix. The structure of the ADP-resistant, R463A mutant of human GDH is identical to native GDH with the exception of the truncated side chain on the pivot helix. Together, these results strongly suggest that ADP activates by facilitating the opening of the catalytic cleft. From alignment of GDH from various sources, it is likely that the antenna evolved in the protista prior to the formation of purine regulatory sites. This suggests that there was some selective advantage of the antenna itself and that animals evolved new functions for GDH through the addition of allosteric regulation.
We report on a rapid simulation method for predicting protein orientation on a surface based on electrostatic interactions. New methods for predicting protein immobilization are needed because of the increasing use of biosensors and protein microarrays, two technologies that use protein immobilization onto a solid support, and because the orientation of an immobilized protein is important for its function. The proposed simulation model is based on the premise that the protein interacts with the electric field generated by the surface, and this interaction defines the orientation of attachment. Results of this model are in agreement with experimental observations of immobilization of mitochondrial creatine kinase and type I hexokinase on biological membranes. The advantages of our method are that it can be applied to any protein with a known structure; it does not require modeling of the surface at atomic resolution and can be run relatively quickly on readily available computing resources. Finally, we also propose an orientation of membrane-bound cytochrome c, a protein for which the membrane orientation has not been unequivocally determined.
• electric double layer
• electrostatic simulations
• orientation flexibility
In this study, we present conformational energies for a molecular mechanical model (Parm99) developed for organic and biological molecules using the restrained electrostatic potential (RESP) approach to derive the partial charges. This approach uses the simple “generic” force field model (Parm94), and attempts to add a minimal number of extra Fourier components to the torsional energies, but doing so only when there is a physical justification. The results are quite encouraging, not only for the 34-molecule set that has been studied by both the highest level ab initiomodel (GVB/LMP2) and experiment, but also for the 55-molecule set for which high-quality experimental data are available. Considering the 55 molecules studied by all the force field models for which there are experimental data, the average absolute errors (AAEs) are 0.28 (this model), 0.52 (MM3), 0.57 (CHARMm [MSI]), and 0.43 kcal/mol (MMFF). For the 34-molecule set, the AAEs of this model versus experiment and ab initio are 0.28 and 0.27 kcal/mol, respectively. This is a lower error than found with MM3 and CHARMm, and is comparable to that found with MMFF (0.31 and 0.22 kcal/mol). We also present two examples of how well the torsional parameters are transferred from the training set to the test set. The absolute errors of molecules in the test set are only slightly larger than in the training set (differences of <0.1 kcal/mol). Therefore, it can be concluded that a simple “generic” force field with a limited number of specific torsional parameters can describe intra- and intermolecular interactions, although all comparison molecules were selected from our 82-compound training set. We also show how this effective two-body model can be extended for use with a nonadditive force field (NAFF), both with and without lone pairs. Without changing the torsional parameters, the use of more accurate charges and polarization leads to an increase in average absolute error compared with experiment, but adjustment of the parameters restores the level of agreement found with the additive model. After reoptimizing the Ψ, Φ torsional parameters in peptides using alanine dipeptide (6 conformational pairs) and alanine tetrapeptide (11 conformational pairs), the new model gives better energies than the Cornell et al. ( J Am Chem Soc 1995, 117, 5179–5197) force field. The average absolute error of this model for high-level ab initio calculation is 0.82 kcal/mol for alanine dipeptide and tetrapeptide as compared with 1.80 kcal/mol for the Cornell et al. model. For nucleosides, the new model also gives improved energies compared with the Cornell et al. model. To optimize force field parameters, we developed a program called parmscan, which can iteratively scan the torsional parameters in a systematic manner and finally obtain the best torsional potentials. Besides the organic molecules in our test set, parmscan was also successful in optimizing the Ψ, Φ torsional parameters in peptides to significantly improve agreement between molecular mechanical and high-level ab initio energies. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1049–1074, 2000
The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg‐yolk, and with beef brain phosphatidylserine was investigated by steady‐state kinetic methods.
The phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids was found to diminish with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipid brought about a rapid, reversible inhibition which remained stable with time for at least 150 min.
The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non‐competitive interaction with K i(app) values varying between (0.9–6.1) × 10 ⁻⁶ M.
The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c , show a higher affinity for the anionic membranes and can dissociate the enzyme · lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid.
Sodium dodecylsulphate causes a time‐dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied.
The relationship of these observations to the structure and function of the enzyme is discussed. It is suggested that their apparent regulation of the enzyme by oestrogens and other small molecules is due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo . The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.
Self-assembled monolayers (SAMs) of 11-mercapto-1-undecanol (TOH) and mixed monolayers of TOH and dodecanethiol (TCH3) were transformed to isocyanate-bearing layers by reaction of the hydroxyl groups in the monolayers and mixed layers with 1,4-phenylene diisocyanate (PDI). PDI reacts with only one of its isocyanate groups, yielding a carbamate (urethane) group; conversion of the hydroxyl groups is nearly quantitative, and the attached phenylene isocyanate moiety is oriented perpendicularly to the surface, as indicated by IR reflection measurements at grazing incidence. The free isocyanate groups readily react under mild conditions with a variety of substances, for example, water, alcohols, and amines. Hence, the conversion of surface hydroxyl groups with PDI offers a versatile and straightforward method to modify surfaces and to facilitate further reactions with additional compounds.
Long-chain alkanethiols (HS(CH2)nX) adsorb from solution onto the surfaces of freshly evaporated copper, silver, and gold films and form oriented monolayers. Both polar and nonpolar tail groups (X) can be accommodated in these adsorptions. Adsorption on all three metals generates self-assembled monolayers (SAMs) exhibiting similar wetting properties. X-ray photoelectron spectroscopy (XPS) data suggest that the omega-terminated n-alkanethiolate monolayers, like those derived from simple alkanethiols, are composed of trans-extended chains having orientations on copper and silver that are closer to the perpendicular to the surface than are those on gold. These observations suggest that variations in the structure of the underlying polymethylene region of these SAMs have little effect on the interfacial free energy of the SAM as manifested by wetting. We have also characterized monolayers ("mixed monolayers") prepared by exposure of all three metals to mixtures of HS(CH2)11OH and HS(CH2)11CH3. On all three, the wettability of the interfaces covers the range between the extremes: theta(a)H2O = approximately 10-degrees and approximately 115-degrees. Values of the advancing contact angle of water can be related to their composition by Cassie's expression. The similarity in wettabilities of these surfaces and the fact that wettability is related to surface composition by a simple linear relationship both argue that CH2OH and CH2CH3 functional groups behave approximately independently at the monolayer-air (water) interface.
The electrostatic free energy of two oppositely charged macroions, suspended in ionic solution is calculated for different distances between the macroions. This is done by using a lattice field theory formulation of the statistical mechanics of Coulomb gas particles of finite size interacting with a fixed charge distribution (J. Chem. Phys. 1995, 102, 4584). In many cases, it is found that, regardless of the size of the mobile ions, the minimum of the free energy is at a separation between the macroions corresponding to a noncontact configuration. Illustrative examples, which emphasize the source of the repulsion at near-touching configurations are presented and discussed.
Mixed self-assembled monolayers (SAMs) composed of a medium length, reactive, n-organothiol (11-mercaptoundecanoic acid) and a short length, unreactive, hydrophobic n-alkylthiol (7-heptanethiol) chemisorbed onto evaporated gold surfaces, were used to study the effect of the microenvironment on the structure and activity of immobilized glucose oxidase (GOX). The mixed SAMs were characterized by X-ray photoelectron spectroscopy, contact angle measurements, and cyclic voltammetry. The derivatization of the SAMs for the covalent attachment and the immobilized enzyme were studied by X-ray photoelectron spectroscopy. Quantitative analysis of the amide I band of the infrared spectra of GOX immobilized onto surfaces at the two attainable extremes of the hydrophilicity range available to us indicated that the percentage of sheet increased with increasing hydrophilicity of the microenvironment. The specific activity of GOX was higher when the enzyme was immobilized onto the hydrophobic microenvironment.
Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713–720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181–1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970–9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three-dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings.
The lateral mobility of eosin isothiocyanate-labeled bovine serum albumin irreversibly adsorbed to poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) surfaces from aqueous solution was measured by a combination of total internal reflection fluorescence and fluorescence recovery after pattern photobleaching techniques. Lateral mobility over distances of several micrometers was probed by photobleaching and monitoring fluorescence with the fringe pattern formed by two intersecting coherent laser beams in total internal reflection. The period of the fringes determined the characteristic length for transport on the surface and was varied by changing the angle of intersection. The dependence of the fluorescence recovery time on the period of the fringes indicates that lateral migration of the adsorbed protein on PMMA is by surface diffusion with D = (1.2 ± 0.3) × 10−9cm2/s, while the extent of fluorescence recovery reveals the coexistence of a mobile population and a population that is apparently immobile over 20 min. For comparison, D = (2.6 ± 0.1) × 10−9cm2/s on PDMS.
A systematic study by site-directed mutagenesis has been conducted on the effector site of phosphofructokinase from Escherichia coli to delineate the role of side chains in binding the allosteric activator, GDP, and inhibitor, PEP, and to search for key residues in the allosteric transtion. Target residues were identified from the crystal structure of the enzyme-nucleoside diphosphate complex. It is found that both activator and inhibitor bind to the same set of amino acid side chains. Deletion of positively charged groups (Arg21, Arg25, Arg54, Arg154, and Lys213 mutated to alanine) weakens binding of both effectors by 2-3 kcal/mol, consistent with the disruption of charged hydrogen bonds. Residue Glu187, which is known from the crystal structure to bind the coordinated Mg2+ ion of GDP, is found to have a unique behavior on mutation and appears to be crucial in triggering the allosteric transition. All other residues mutated simply weaken binding of both PEP and GDP in a parallel manner. However, mutation of Glu----Ala187 reverses the roles of GDP and PEP, causing GDP to become an allosteric inhibitor and PEP an activator. Mutation of Glu----Gln187 has only a small effect on the binding of PEP, and both PEP and GDP are inhibitors. Studies are described in which mutations in different subunits of a tetrameric complex complement each other. The effector site is composed of residues from two subunits. In particular, Arg21 and Lys213 in each site are from different subunits. Mutations of either one of these residues abolishes activation by GDP of the homotetramer.(ABSTRACT TRUNCATED AT 250 WORDS)
Initial rate kinetic studies and reduced coenzyme binding studies with bovine glutamate dehydrogenase have shown that an enzyme-NAD(P)H-glutamate abortive complex is a major participant in the overall enzyme-catalyzed reaction. The allosteric regulator ADP is shown to activate the enzyme by destabilizing this abortive complex. Destabilization of this abortive complex with concomitant activation is also achieved by the ethoxyformylation of a single histidine residue per subunit in the hexameric enzyme. ADP, in addition to its activatory effects, is shown to (a) remove the nonlinearity from the Lineweaver-Burk plots which is attributable to negative cooperativity and (b) inhibit the enzyme as a competitive inhibitor with respect to coenzyme under the appropriate conditions. Ethoxyformylation with diethyl pyrocarbonate, which mimics the activatory effects of ADP, has no effect on the negative cooperativity shown by this enzyme. A model for the action of ADP is proposed in which ADP activates glutamate dehydrogenase by binding to a regulatory binding site and blocks the negative cooperativity by mimicking the natural coenzyme at the active site of the enzyme.
The surface diffusion rate of bacterial cellulases from Cellulomonas fimi on cellulose was quantified using fluorescence recovery after photobleaching analysis. Studies were performed on an exo-β-1–4-glycanase
(Cex), an endo-β-1–4-glucanase (CenA), and their respective isolated cellulose-binding domains (CBDs). Although these cellulose-binding
domains bind irreversibly to microcrystalline cellulose, greater than 70% of bound molecules are mobile on the cellulose surface.
Surface diffusion rates are dependent on surface coverage and range from a low of 2 × 10−11 to a maximum of 1.2 × 10−10 cm2/s. The fraction of mobile molecules increases only slightly with increasing fractional surface coverage density. Results
demonstrate that the packing of C. fimicellulases and their isolated binding domains onto the cellulose surface is a dynamic process. This suggests that the exclusion
of potential CBD binding sites on the cellulose due to steric effects of neighboring bound CBDs may not fully explain the
apparent negative cooperativity exhibited in CBD adsorption isotherms. Comparison with the kinetics of cellulase hydrolysis
of crystalline substrate suggests that surface diffusion rates do not limit cellulase activity.
The binding of Src to phospholipid membranes requires both hydrophobic insertion of its myristate into the hydrocarbon interior of the membrane and nonspecific electrostatic interaction of its N-terminal cluster of basic residues with acidic phospholipids. We provide a theoretical description of the electrostatic partitioning of Src onto phospholipid membranes. Specifically, we use molecular models to represent a nonmyristoylated peptide corresponding to residues 2-19 of Src [nonmyr-Src(2-19); GSSKSKPKDPSQRRRSLE-NH2] and a phospholipid bilayer, calculate the electrostatic interaction by solving the nonlinear Poisson-Boltzmann equation, and predict the molar partition coefficient using statistical thermodynamics. The theoretical predictions agree with experimental data obtained by measuring the partitioning of nonmyr-Src(2-19) onto phospholipid vesicles: membrane binding increases as the mole percent of acidic lipid in the vesicles is increased, the ionic strength of the solution is decreased, or the net positive charge of the peptide is increased. The theoretical model also correctly predicts the measured partitioning of the myristoylated peptide, myr-Src(2-19); for example, adding 33% acidic lipid to electrically neutral vesicles increases the partitioning of myr-Src(2-19) 100-fold. Phosphorylating either serine 12 (by protein kinase C) or serine 17 (by cAMP-dependent protein kinase) decreases the partitioning of myr-Src(2-19) onto vesicles containing acidic lipid 10-fold. We investigated the effect of phosphorylation on the localization of Src to biological membranes by expressing fusion constructs of Src's N terminus with a soluble carrier protein in COS-1 cells; phosphorylation produces a small shift in the distribution of the Src chimeras from the plasma membrane to the cytosol.
Bovine glutamate dehydrogenase (boGDH) is a homohexameric, mitochondrial enzyme that reversibly catalyzes the oxidative deamination of L-glutamate to 2-oxoglutarate using either NADP(H) or NAD(H) with comparable efficacy. GDH represents a key enzymatic link between catabolic and biosynthetic pathways, and is therefore ubiquitous in both higher and lower organisms. Only mammalian GDH exhibits strong negative cooperativity with respect to the coenzyme, however, and is regulated by a large number of allosteric effectors.
The atomic structure of boGDH in complex with NADH, glutamate, and the allosteric inhibitor GTP has been determined to 2.8 A resolution. The major difference between the bacterial and bovine GDH structures is the presence of an additional 'antenna' in boGDH that protrudes from each trimer, twisting counterclockwise along the threefold axis. NADH and glutamate are clearly observed in the active site, but the contacts differ slightly from those observed in Clostridium symbiosum GDH. A second, inhibitory NADH molecule lies buried in the core of the hexamer. Finally, two GTP molecules bind near the hinge region connecting the NAD(+)- and glutamate-binding domains.
We propose that the antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation. GTP and NADH inhibit GDH by keeping the catalytic cleft in a closed conformation. In contrast, ADP probably binds to the back of the NAD(+)-binding domain and activates the enzyme by keeping the catalytic cleft open. Extensive contacts between antennae within the crystal lattice may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix.
The interaction of a ligand with a protein occurs at a local site (the binding site) and involves only a few residues; however, the effects of that interaction are often propagated to remote locations. The chain of events initiated by binding provides the basis for fundamental biological phenomena such as allosterism, signal transduction, and structural-stability modification. In this paper, a structure-based statistical thermodynamic approach is presented and used to predict the propagation of the stabilization effects triggered by the binding of the monoclonal antibody D1.3 to hen egg white lysozyme. Previously, Williams et al. [Williams, D. C., Benjamin, D. C., Poljak, R. J. & Rule, G. S. (1996) J. Mol. Biol. 257, 866-876] showed that the binding of this antibody affects the stability of hen egg white lysozyme and that the binding effects propagate to a selected number of residues at remote locations from the binding epitope. In this paper, we show that this phenomenon can be predicted from structure. The formalism presented here permits the identification of the structural path followed by cooperative interactions that originate at the binding site. It is shown that an important condition for the propagation of binding effects to distal regions is the presence of a significant fraction of residues with low structural stability in the uncomplexed binding site. A survey of protein structures indicates that many binding sites have a dual character and are defined by regions of high and low structural stabilities. The low-stability regions might be involved in the transmission of binding information to other regions in the protein.
Glutamate dehydrogenase is found in all organisms and catalyses the oxidative deamination of l-glutamate to 2-oxoglutarate. However, only animal GDH utilizes both NAD(H) or NADP(H) with comparable efficacy and exhibits a complex pattern of allosteric inhibition by a wide variety of small molecules. The major allosteric inhibitors are GTP and NADH and the two main allosteric activators are ADP and NAD(+). The structures presented here have refined and modified the previous structural model of allosteric regulation inferred from the original boGDH.NADH.GLU.GTP complex. The boGDH.NAD(+).alpha-KG complex structure clearly demonstrates that the second coenzyme-binding site lies directly under the "pivot helix" of the NAD(+) binding domain. In this complex, phosphates are observed to occupy the inhibitory GTP site and may be responsible for the previously observed structural stabilization by polyanions. The boGDH.NADPH.GLU.GTP complex shows the location of the additional phosphate on the active site coenzyme molecule and the GTP molecule bound to the GTP inhibitory site. As expected, since NADPH does not bind well to the second coenzyme site, no evidence of a bound molecule is observed at the second coenzyme site under the pivot helix. Therefore, these results suggest that the inhibitory GTP site is as previously identified. However, ADP, NAD(+), and NADH all bind under the pivot helix, but a second GTP molecule does not. Kinetic analysis of a hyperinsulinism/hyperammonemia mutant strongly suggests that ATP can inhibit the reaction by binding to the GTP site. Finally, the fact that NADH, NAD(+), and ADP all bind to the same site requires a re-analysis of the previous models for NADH inhibition.
During the course of their biological function, proteins undergo different types of structural rearrangements ranging from local to large-scale conformational changes. These changes are usually triggered by their interactions with small-molecular-weight ligands or other macromolecules. Because binding interactions occur at specific sites and involve only a small number of residues, a chain of cooperative interactions is necessary for the propagation of binding signals to distal locations within the protein structure. This process requires an uneven structural distribution of protein stability and cooperativity as revealed by NMR-detected hydrogen/deuterium exchange experiments under native conditions. The distribution of stabilizing interactions does not only provide the architectural foundation to the three-dimensional structure of a protein, but it also provides the required framework for functional cooperativity. In this review, the statistical thermodynamic linkage between protein stability, functional cooperativity, and ligand binding is discussed.
The structure of human glutamate dehydrogenase (GDH) has been determined in the absence of active site and regulatory ligands. Compared to the structures of bovine GDH that were complexed with coenzyme and substrate, the NAD binding domain is rotated away from the glutamate-binding domain. The electron density of this domain is more disordered the further it is from the pivot helix. Mass spectrometry results suggest that this is likely due to the apo form being more dynamic than the closed form. The antenna undergoes significant conformational changes as the catalytic cleft opens. The ascending helix in the antenna moves in a clockwise manner and the helix in the descending strand contracts in a manner akin to the relaxation of an extended spring. A number of spontaneous mutations in this antenna region cause the hyperinsulinism/hyperammonemia syndrome by decreasing GDH sensitivity to the inhibitor, GTP. Since these residues do not directly contact the bound GTP, the conformational changes in the antenna are apparently crucial to GTP inhibition. In the open conformation, the GTP binding site is distorted such that it can no longer bind GTP. In contrast, ADP binding benefits by the opening of the catalytic cleft since R463 on the pivot helix is pushed into contact distance with the beta-phosphate of ADP. These results support the previous proposal that purines regulate GDH activity by altering the dynamics of the NAD binding domain. Finally, a possible structural mechanism for negative cooperativity is presented.
The biomolecular conformational changes often associated with allostery are, by definition, dynamic processes. Recent publications have disclosed the role of pre-existing equilibria of conformational substates in this process. In addition, the role of dynamics as an entropic carrier of free energy of allostery has been investigated. Recent work thus shows that dynamics is pivotal to allostery, and that it constitutes much more than just the move from the 'T'-state to the 'R'-state. Emerging computational studies have described the actual pathways of allosteric change.
Continuum solvation models, such as Poisson–Boltzmann and Generalized Born methods, have become increasingly popular tools
for investigating the influence of electrostatics on biomolecular structure, energetics and dynamics. However, the use of
such methods requires accurate and complete structural data as well as force field parameters such as atomic charges and radii.
Unfortunately, the limiting step in continuum electrostatics calculations is often the addition of missing atomic coordinates
to molecular structures from the Protein Data Bank and the assignment of parameters to biomolecular structures. To address
this problem, we have developed the PDB2PQR web service (http://agave.wustl.edu/pdb2pqr/). This server automates many of the common tasks of preparing structures for continuum electrostatics calculations, including
adding a limited number of missing heavy atoms to biomolecular structures, estimating titration states and protonating biomolecules
in a manner consistent with favorable hydrogen bonding, assigning charge and radius parameters from a variety of force fields,
and finally generating ‘PQR’ output compatible with several popular computational biology packages. This service is intended
to facilitate the setup and execution of electrostatics calculations for both experts and non-experts and thereby broaden
the accessibility to the biological community of continuum electrostatics analyses of biomolecular systems.
Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.
A combination of thirty-two 10-ns-scale molecular dynamics simulations were used to explore the coupling between conformational transition and phosphorylation in the bacteria chemotaxis Y protein (CheY), as a simple but representative example of protein allostery. Results from these simulations support an activation mechanism in which the beta4-alpha4 loop, at least partially, gates the isomerization of Tyr106. The roles of phosphorylation and the conserved Thr87 are deemed indirect in that they stabilize the active configuration of the beta4-alpha4 loop. The indirect role of the activation event (phosphorylation) and/or conserved residues in stabilizing, rather than causing, specific conformational transition is likely a feature in many signaling systems. The current analysis of CheY also helps to make clear that neither the "old" (induced fit) nor the "new" (population shift) views for protein allostery are complete, because they emphasize the kinetic (mechanistic) and thermodynamic aspects of allosteric transitions, respectively. In this regard, an issue that warrants further analysis concerns the interplay of concerted collective motion and sequential local structural changes in modulating cooperativity between distant sites in biomolecules.
Enzymes adsorbed on palmityl-substituted Sepharose 4B by hydrophobic interactions have been used in reactor-type experiments. Results presented on immobilized glutamate dehydrogenase, trypsin, alpha-chymotrypsin, and amyloglucosidase indicate possible potential of the method for continuous catalytic operations. Glutamate dehydrogenase used as a model allosteric enzyme was found to retain its allosteric properties after binding to the absorbent in the form of column or suspension. Thermal stabilities of glutamate dehydrogenase and alpha-chymotrypsin were significantly decreased upon adsorption, while that of trypsin was apparently unaltered. Results are discussed in terms of specific interactions involving palmityl residues present on the matrix. Relevance of these observations to in vivo processes are also discussed.
Flexibility of a Model Protein upon Immobilization
- Bigdeli
Bigdeli et al.: Flexibility of a Model Protein upon Immobilization