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PubMed2006

Characterization of a new radiochromic three-dimensional dosimeter.

The development of intensity-modulated radiotherapy (IMRT) has created a clear need for a dosimeter that can accurately and conveniently measure dose distributions in three dimensions to assure treatment quality. PRESAGE is a new three dimensional (3D) dosimetry material consisting of an optically clear polyurethane matrix, containing a leuco dye that exhibits a radiochromic response when exposed to ionizing radiation. A number of potential advantages accrue over other gel dosimeters, including insensitivity to oxygen, radiation induced light absorption contrast rather than scattering contrast, and a solid texture amenable to machining to a variety of shapes and sizes without the requirement of an external container. In this paper, we introduce an efficient method to investigate the basic properties of a 3D dosimetry material that exhibits an optical dose response. The method is applied here to study the key aspects of the optical dose response of PRESAGE: linearity, dose rate dependency, reproducibility, stability, spectral changes in absorption, and temperature effects. PRESAGE was prepared in 1 x 1 x 4.5 cm3 optical cuvettes for convenience and was irradiated by both photon and electron beams to different doses, dose rates, and energies. Longer PRESAGE columns (2 x 2 x 13 cm3) were formed without an external container, for measurements of photon and high energy electron depth-dose curves. A linear optical scanning technique was used to detect the depth distribution of radiation induced optical density (OD) change along the PRESAGE columns and cuvettes. Measured depth-OD curves were compared with percent depth dose (PDD). Results indicate that PRESAGE has a linear optical response to radiation dose (with a root mean square error of -1%), little dependency on dose rate (-2%), high intrabatch reproducibility (< 2%), and can be stable (-2%) during 2 hours to 2 days post irradiation. Accurate PRESAGE dosimetry requires temperature control within 1 degrees C. Variations in the PRESAGE formulation yield corresponding variations in sensitivity, stability, and density. CT numbers in the range 100-470 were observed. In conclusion, the small volume studies presented here indicate PRESAGE to be a promising, versatile, and practical new dosimetry material with applicability for radiation therapy.

Dose-Response Relationship, Radiation↗
PubMed2000

Plasticity and steric strain in a parallel beta-helix: rational mutations in the P22 tailspike protein.

By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89-101.

Bacteriophage P22↗
PubMed2000

EPR studies of chromium(V) intermediates generated via reduction of chromium(VI) by DOPA and related catecholamines: potential role for oxidized amino acids in chromium-induced cancers.

The reductions of K2Cr2O7 by catecholamines, DOPA, DOPA-beta,beta-d2, N-acetyl-DOPA, alpha-methyl-DOPA, dopamine, adrenaline, noradrenaline, catechol, 1,2-dihydroxybenzoic acid (DHBA), and 4-tert-butylcatechol (TBC), produce a number of Cr(V) electron paramagnetic resonance (EPR) signals. These species are of interest in relation to the potential role of oxidized proteins and amino acids in Cr-induced cancers. With excess organic ligand, all of the substrates yield Cr species with signals at g(iso) approximately 1.972 (Aiso(53Cr) > 23.9 x 10(-4) cm(-1)). These are similar to signals reported previously but have been reassigned as octahedral Cr(V) species with mixed catechol-derived ligands, [CrV(semiquinone)2(catecholate)]+. Experiments with excess K2Cr2O7 show complex behavior with the catecholamines and TBC. Several weak Cr(V) signals are detected after mixing, and the spectra evolve over time to yield relatively stable substrate-dependent signals at g(iso) approximately 1.980. These signals have been attributed to [Cr(O)L2](L = diolato) species, in which the Cr is coordinated to two cyclized catecholamine ligands and an oxo ligand. Isotopic labeling studies with DOPA (ring or side chain deuteration or enrichment with 15N), and simulation of the signals, show that the superhyperfine couplings originate from the side chain protons, confirming that the catecholamine ligands are cyclized. At pH 3.5, a major short-lived EPR signal is observed for many of the substrates at g(iso) approximately 1.969, but the species responsible for this signal was not identified. Several other minor Cr signals are detected, which are attributed (by comparison with isoelectronic V(IV) species) to Cr(V) complexes coordinated by a single catecholamine ligand (and auxiliary ligands e.g. H2O), or to [Cr(O)L2]- (L = diolato) species with a sixth ligand (e.g. H2O). Addition of catalase or deoxygenation of the solutions did not affect the main EPR signals. When the substrates were in excess (pH > 4.5), primary and secondary (cyclized) semiquinones were also detected. Semiquinone stabilization by Zn(II) complexation yielded stronger EPR signals (g(iso) approximately 2.004).

Amino Acids↗
PubMed1965

The interaction of collagen and acid mucopolysaccharides. A model for connective tissue.

1. The interaction of acid mucopolysaccharides of connective tissue with solubilized collagen of native, or near-native, structure was investigated by free solution electrophoresis at pH7.0. 2. Complex-formation was detected by the appearance of a third peak in the ascending limb only, indicating reversible association. 3. Complex-formation was destroyed by prior heating of solubilized collagen, indicating a probable requirement for high molecular weight or internal structure of the protein. 4. Hyaluronate and chondroitin sulphate of mol.wt. 50000 gave complexes with soluble collagen at I 0.4, whereas heparin and chondroitin sulphate of mol.wt. 15000-18000 did not. All mucopolysaccharides yielded complexes at I 0.1. The stability of the complex appears mainly dependent on electrostatic forces and is increased with increase in chain length of the polysaccharide. 5. Solubilized collagen interacted to yield gels with the ;native' chondroitin sulphate-protein macro-molecule from cartilage. 6. A schematic model for the interaction of collagen and chondroitin sulphate-protein macromolecules shows parallel-ordered interaction of collagen fibrils with chondroitin sulphate side chains of the chondroitin sulphate-protein macromolecule. The biological implications of this model are discussed, particularly in relation to the ordered structures and the ionic-network properties of the intercellular components of connective tissue.

Animals↗
PubMed2003

Effect of catalysis on the stability of metallic nanoparticles: Suzuki reaction catalyzed by PVP-palladium nanoparticles.

The small size of nanoparticles makes them attractive in catalysis due to their large surface-to-volume ratio. However, being small raises questions about their stability in the harsh chemical environment in which these nanoparticles find themselves during their catalytic function. In the present work, we studied the Suzuki reaction between phenylboronic acid and iodobenzene catalyzed by PVP-Pd nanoparticles to investigate the effect of catalysis, recycling, and the different individual chemicals on the stability and catalytic activity of the nanoparticles during this harsh reaction. The stability of the nanoparticles to the different perturbations is assessed using TEM, and the changes in the catalytic activity are assessed using HPLC analysis of the product yield. It was found that the process of refluxing the nanoparticles for 12 h during the Suzuki catalytic reaction increases the average size and the width of the distribution of the nanoparticles. This was attributed to Ostwald ripening in which the small nanoparticles dissolve to form larger nanoparticles. The kinetics of the change in the nanoparticle size during the 12 h period show that the nanoparticles increase in size during the beginning of the reaction and level off toward the end of the first cycle. When the nanoparticles are recycled for the second cycle, the average size decreases. This could be due to the larger nanoparticles aggregating and precipitating out of solution. This process could also explain the observed loss of the catalytic efficiency of the nanoparticles during the second cycle. It is also found that the addition of biphenyl to the reaction mixture results in it poisoning the active sites and giving rise to a low product yield. The addition of excess PVP stabilizer to the reaction mixture seems to lead to the stability of the nanoparticle surface and size, perhaps due to the inhibition of the Ostwald ripening process. This also decreases the catalytic efficiency of the nanoparticles due to capping of the nanoparticle surface. The addition of phenylboronic acid is found to lead to the stability of the size distribution as it binds to the particle surface through the O(-) of the OH group and acts as a stabilizer. Iodobenzene is found to have no effect and thus probably does not bind strongly to the surface during the catalytic process. These two results might have an implication on the catalytic mechanism of this reaction.

Journal Article↗
PubMed1994

Interdomain loops and conformational changes of glutamate synthase as detected by limited proteolysis.

Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, was subjected to limited proteolysis using trypsin and chymotrypsin, in the absence or presence of its substrates or their analogs. Time-dependent degradation of glutamate synthase alpha and beta subunits, to yield several fragments of different stability, was observed, the alpha subunit being more sensitive than the beta to proteolytic attack. The main sites of proteolytic cleavage were determined by densitometric analysis of the electrophoretic patterns obtained under denaturing conditions and by N-terminal sequencing of the major proteolytic products. These analyses showed that most of the peptide bonds sensitive to the proteases are clustered in two regions of the alpha subunit, outside the proposed substrate and cofactor binding regions of glutamate synthase [Pelanda, R., Vanoni, M. A., Perego, M., Piubelli, L., Galizzi, A., Curti, B. & Zanetti, G. (1993) J. Biol. Chem. 268, 3099-3106]. Therefore, these protease-sensitive sites can be identified as flexible loops, exposed to solvent, connecting adjacent domains of the protein. The presence of the enzyme substrates or their analogs caused significant changes in the proteolytic patterns. NADP+ protected the C-terminal region of glutamate synthase beta subunit from tryptic cleavage, supporting the proposal that it contains the pyridine-nucleotide-binding site. Furthermore, NADP+, and to a lesser extent the glutamine analog L-methionine sulfone, which binds presumably to the N-terminal region of the alpha subunit, altered the sensitivity to proteolysis of the sites of the alpha subunit proposed to be part of links between domains of glutamate synthase. These results show that long-range conformational changes of glutamate synthase occur on binding of its substrates. The study of several NADPH-dependent diaphorase activities of glutamate synthase was also undertaken in order to test if proteolytic fragments of the enzyme retained their ability to transfer electrons from NADPH to synthetic electron acceptors. Although proteolysis yielded partial loss of all enzyme NADPH-dependent reactions, the kinetic analysis showed that the rates of reduction of iodonitrotetrazolium, ferricyanide and dichlorophenolindophenol were at least twofold faster than the rate of the physiological glutamate synthase reaction. These results indicate that enzyme reduction and intramolecular electron transfer are not rate limiting during catalysis of the physiological glutamate synthase reaction.

2,6-Dichloroindophenol↗
PubMed2003

Lentivector-mediated transfer of Bmi-1 and telomerase in muscle satellite cells yields a duchenne myoblast cell line with long-term genotypic and phenotypic stability.

Conditionally immortalized human cells are valuable substrates for basic biologic studies, as well as for the production of specific proteins and for the creation of bioartificial organs. We previously demonstrated that the lentivector-mediated transduction of immortalizing genes into human primary cells is an efficient method for obtaining such cell lines. Here, we used human muscle satellite cells as model targets to examine the impact of the transduced genes on the genotypic and phenotypic characteristics of the immortalized cells. The most commonly used immortalizing gene, the SV40 large T antigen (T-Ag), was extremely efficient at inducing the continuous growth of primary myoblasts, but the resulting cells rapidly accumulated major chromosomal aberrations and exhibited profound phenotypic changes. In contrast, the constitutive expression of telomerase and Bmi-1 in satellite cells from a control individual and from a patient suffering from Duchenne's muscular dystrophy yielded cell lines that remained diploid and conserved their growth factor dependence for proliferation. However, despite the absence of detectable cytogenetic abnormalities, clones derived from satellite cells of a control individual exhibited a differentiation block in vitro. In contrast, a Duchenne-derived cell line exhibited all the phenotypic characteristics of its primary parent, including an ability to differentiate fully into myotubes when placed in proper culture conditions. This cell line should constitute a useful reagent for a wide range of studies aimed at this disease.

Adolescent↗
PubMed1984

Effect of the lyotropic series of anions on denaturation and renaturation of 20 beta-hydroxysteroid dehydrogenase.

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denatured enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the activation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1-2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.

20-Hydroxysteroid Dehydrogenases↗
PubMed1998

Investigation of biosynthesis of human lymphotoxin in cells of recombinant Escherichia coli strain.

The effect of cultivation conditions on the biosynthesis of human lymphotoxin in recombinant Escherichia coli SG20050/pLT21 strain was studied. Cells of the producing strain were grown in Luria broth containing chloramphenicol. The highest biomass yield of the recombinant strain and plasmid DNA stability were observed under these conditions. To enhance the level of lymphotoxin production an inoculate containing freshly obtained or frozen with glycerol transformants of the producing strain were used, and the cultivation process was performed at 32 degrees C. As a result, lymphotoxin was synthesized in a soluble form without the formation of inclusion bodies. A study of the protein synthesis dynamics during the cultivation of E. coli at 32 degrees C showed that the highest lymphotoxin activity was observed during the exponential growth phase, being maximal at the end of the exponential phase and at the beginning of the stationary phase. The set of indicated methods allowed us to maximize and stabilize the production of lymphotoxin in a biologically active form with a final yield of 18-20% from cell protein.

Cell Division↗
PubMed1996

Antibodies released from immunoadsorbents: effect of support, activation and elution conditions.

Immunoadsorption is an application of affinity chromatography, as a therapeutic method to specifically deplete biological fluids such as blood plasma from proteins in excess, or to extract a biomolecule from a complex mixture. However, the leakage of small amounts of antibodies covalently immobilized on the support hampers the practical use of this method. In fact, these released antibodies contaminate the purified proteins or depleted media and, when they are of animal nature, they may lead to immunization of patients, or cause an anaphylactic shock when a clinical use is concerned. It is therefore of prime importance that the immunoadsorbents exhibit a satisfactory stability over the whole range of chemical and biochemical conditions involved during their clinical handling. To determine optimal conditions for the preparation of stable immunoadsorbents designed to remove selectively Low Density Lipoproteins (LDLs) from the plasma of patients affected by familial hypercholesterolemia, various immunoadsorbents were prepared by covalent immobilization of goat anti-apolipoprotein B polyclonal antibodies on different supports (Sepharose CL-4B, Sepharose 6 Fast Flow, Sphérodex and Fractogel) previously activated by various chemical reagents (cyanogen bromide, divinyl sulphone, tresyl chloride and trichloro-s-triazine). Their adsorption capacity, specificity, stability and the amount of immobilized antibodies were compared in terms of the activation method and the support used. It turns out that the immunoadsorbents prepared with Sepharose 6 Fast Flow lead to optimal yield of coupling, adsorption capacity, and an excellent stability at neutral pH. TC-activated-Fractogel turns out as well to afford an excellent coupling yield, a good adsorption capacity and an optimal stability in the whole pH range tested.

Adsorption↗
PubMed1998

Three novel type I collagen mutations in osteogenesis imperfecta type IV probands are associated with discrepancies between electrophoretic migration of osteoblast and fibroblast collagen.

In three cases of type IV osteogenesis imperfecta (OI), we identified unique point mutations in type I collagen alpha1(I) cDNA. In two cases, the appearance of dimers indicated the presence of cysteine substitutions in the alpha1(I) protein chain. Cyanogen bromide digestion localized these cross-links to CB8 and 3, respectively. In the third case, the overmodification pattern of the CNBr peptides was compatible with a substitution in the aa 123-402 region of either type I collagen chain. We identified a unique point mutation in each proband, which resulted in substitutions for glycine residues in a 300-aa region of the alpha1(I) helix, specifically, Gly to Ala at codon 220 (GGT-->GCT), Gly to Cys at codon 349 (GGT-->TGT) and Gly to Cys at codon 523 (GGT-->TGT). We compared each proband's fibroblast and osteoblast collagen directly, as well as with fibroblast and osteoblast controls. For all cases, the OI osteoblast collagen was more electrophoretically delayed than OI fibroblast collagen. In the patient with G349C, OI fibroblast and osteoblast collagen synthesized in the presence of alpha,alpha'-dipyridyl co-migrated on gels, demonstrating that the electrophoretic discrepancy resulted from differences in post-translational modification. Melting temperature curves for stability of the collagen helix yielded an identical Tm for control fibroblast and osteoblast collagen (41.2 degrees C). By contrast, for collagen with the gly349-->cys substitution, the Tm of the fibroblast collagen was 1 degree C lower than the Tm of the osteoblast collagen. These data indicate that the metabolism of mutant collagen might be cell-specific and has significant implications for understanding the phenotype/genotype correlations and the pathophysiology of OI.

Adolescent↗
PubMed1996

Five modified numerical deconvolution methods for biopharmaceutics and pharmacokinetics studies.

Four improved finite-difference numerical deconvolution methods and one nonlinear regression numerical deconvolution method are proposed and implemented using IMSL/IDLTM. These five numerical deconvolution methods are evaluated using simulated data generated with and without added noise under six different dosing cases. Comparisons between these methods are made in terms of the superimposability of the calculated cumulative amount of drug released or absorbed-time profiles with the theoretical data. The results indicate that the proposed fixed step number equal step length numerical deconvolution method is simple and accurate and therefore is appropriate for pharmacokinetic and biopharmaceutic studies. When an analytic function is legitimate to represent the drug input rate, the nonlinear regression numerical deconvolution method will yield enhanced numerical accuracy and stability.

Absorption↗
PubMed1984

Assessment of body elimination attitude.

Constructed a 26-item Likert format Body Elimination Attitude Scale with good internal consistency and temporal stability (N = 60). Factor analysis yielded seven factors labeled Fecal Smell, Personal Hygiene, Sight, Dirty Hair, Animal Feces, Mucous-like Discharge, and Sound. Both the prominence of items that pertained to defecation and the positive association of the Scale with a measure of obsessive personality-anal character were viewed as consistent with psychoanalytic theory. Correlations with psychometric instruments revealed greater body elimination disgust to be associated with psychopathology. Youth, lesser education, having fewer children, being female, and lesser income also were associated with greater disgust.

Adolescent↗
PubMed1976

Immobilization of enzymes based on hydrophobic interaction. I. Preparation and properties of a beta-amylase adsorbate.

Sweet potato beta-amylase (alpha-1,4 glucan maltohydrolase, EC 3.2.1.2) was immobilized through adsorption onto an agrose gel to which nonpolar side chains had been introduced via ether bridges. The adsorbent showed evidence of saturation at an enzyme content of 35 mg per milliliter of packed gel. The adsorption was rapid and yielded a product whose operational stability depended on the initial content of beta-amylase. Activity leakage was low. The relative activity of immobilized enzyme was inversely related to the amount of enzyme adsorbed to a given gel volume, having a maximal value of around 50% at low enzyme contents.

Adsorption↗
PubMed2005

A short and concise asymmetric synthesis of hamigeran B.

The interesting biological properties of the hamigerans wherein hamigeran B is a potent antiviral agent with low cytotoxicity to host cells make these deceptively simple looking structures challenging synthetic targets. A strategy to hamigeran B evolved wherein the three contiguous stereocenters are established ultimately from a Pd catalyzed asymmetric allylic alkylation (AAA). The latter involves an asymmetric allylation of a non-stabilized ketone enolate in 77 % yield and 93 % ee. By using this process, (S)-5-allyl-2-isopropyl-5-methyl-1-trifluoromethanesulfonyloxycyclopentene becomes available in four steps from 2-methylcyclopentanone. Introduction of the aryl unit by cross-coupling proceeded intermolecularly but failed intramolecularly. On the other hand, reductive removal of the triflate permitted a Heck reaction to effect intramolecular introduction of the aryl ring. The unusual conformational properties of this molecular architecture are revealed by the regioselectivity of the beta-hydrogen elimination in the Heck reaction and the diastereoselectivity of the reduction establishing the stereochemistry of the carbon bearing the isopropyl group. The successful route consists of 15 steps from 2-methylcyclopentanone and dimethylorcinol illustrating the efficiency of the route based upon the Pd AAA.

Hydrogenation↗
PubMed2007

Photosynthetic reaction center mimicry of a "special pair" dimer linked to electron acceptors by a supramolecular approach: self-assembled cofacial zinc porphyrin dimer complexed with fullerene(s).

Biomimetic bacterial photosynthetic reaction center complexes have been constructed using well-defined self-assembled supramolecular approaches. The "special pair" donor, a cofacial porphyrin dimer, was formed via potassium ion induced dimerization of meso-(benzo-[15]crown-5)porphyrinatozinc. The dimer was subsequently self-assembled with functionalized fullerenes via axial coordination and crown ether-alkyl ammonium cation complexation to form the donor-acceptor pairs, mimicking the noncovalently bound entities of the photosynthetic reaction center. The adopted self-assembly methodology yielded supramolecular complexes of higher stability, with defined geometry and orientation. Efficient forward electron transfer from the singlet excited zinc porphyrin dimer to the fullerene entity and relatively slow reverse electron transfer, important steps in the photosynthetic light energy conversion have been achieved in these novel biomimetic model systems.

Dimerization↗
PubMed1993

Detection system for membrane immunoassay based on the trapping of a highly colored intermediate of the peroxidase reaction.

The oxidation of ortho-dianisidine by membrane bound horseradish peroxidase in the presence of sodium dextran sulfate affords a dark-green insoluble product, identified as an unstable meriquinone intermediate previously reported in literature. Cationic and unsubstituted dextrans do not stabilize the intermediate. The highest yield of the intermediate is observed at pH 4.0-5.0 and concentration of dextran sulfate ca. 0.5%. New highly sensitive detection system for peroxidase has been developed on the basis of ortho-dianisidine oxidation to the meriquinone intermediate in the presence of sodium dextran sulfate. Under certain conditions, with lowered sodium dextran sulfate concentrations, a progressive further oxidation of the green intermediate to the yellow-brown final product is observed on passing to higher enzyme concentrations. This finding opens a possibility to develop a detection system in which the color of the mixture of reaction products serves as a measure of the enzyme concentration.

Chemistry Techniques, Analytical↗
PubMed2000

Molecular dynamics simulations highlight mobile regions in proteins: A novel suggestion for converting a murine V(H) domain into a more tractable species.

The V(H) region of the murine antibody 1F7 has been identified as a single-domain chorismate mutase, but a tendency to denature and aggregate has hampered its biochemical characterization. Standard mutagenesis approaches targeting antibody chain dimerization areas have been exhausted. We describe a new approach to the problem, where we use molecular dynamics (MD) simulations to find the differences between the untractable protein and the known soluble V(H) domain from a llama antibody. MD simulations of proteins yield information on the relative stability and fluctuations of parts of the proteins. By comparing simulation results of two related proteins their differences in stability and fluctuations can be analyzed and may suggest mutations aimed at (de)stabilization of one of the two proteins. For the mouse versus llama simulations, this approach highlights an untried area in the protein which shows increased fluctuations. The replacement of this eight-residue segment with the corresponding llama sequence gave a chimeric mutant which shows significantly decreased fluctuations. We see this as a general scheme to generate suggestions for mutagenesis experiments, not only obviously generalizable to other immunoglobulin domains, but to other protein systems as well.

Amino Acid Sequence↗