Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

Cholesterol‐β1AR interaction versus cholesterol‐β2AR interaction

Two 8‐µs all‐atom molecular dynamics simulations have been performed on the two highly homologous G protein‐coupled receptor (GPCR) subtypes, β₁‐ and β₂‐adrenergic receptors, which were embedded in a lipid bilayer with randomly dispersed cholesterol molecules. During the simulations, cholesterol molecules accumulate to different surface regions of the two receptors, suggesting the subtype specificity of cholesterol–β‐adrenergic receptor interaction and providing some clues to the physiological difference of the two subtypes. Meanwhile, comparison between the two receptors in interacting with cholesterols shed some new light on general determinants of cholesterol binding to GPCRs. Our results indicate that although the concave surface, charged residues and aromatic residues are important, neither of these stabilizing factors is indispensable for a cholesterol interaction site. Different combinations of these factors lead to the diversified binding modes of cholesterol binding to the receptors. Our long‐time simulations, for the first time, revealed the pathway of a cholesterol molecule entering the consensus cholesterol motif (CCM) site, and the binding process of cholesterol to CCM is accompanied by a side chain flipping of the conserved Trp4.50. Moreover, the simulation results suggest that the I‐/V‐/L‐rich region on the extracellular parts of helix 6 might be an alternatively conserved cholesterol‐binding site for the class‐A GPCRs. Proteins 2014; 82:760–770. © 2013 Wiley Periodicals, Inc.     

Comparison of marmoset and human FSH using synthetic peptides of the β‐subunit L2 loop region and anti‐peptide antibodies

Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH–FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti‐peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH β‐subunit (L2β) was found to be different between marmoset and human. Hence, peptides corresponding to region 32–50 of marmoset and human L2β loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone–receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross‐species peptide and FSH in ELISA. The anti‐peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2β loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH–FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

β‐Hairpin stabilization in a 28‐residue peptide derived from the β‐subunit sequence of human chorionic gonadotropin hormone

The β‐subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin‐like fragments. To investigate the role of β‐hairpin formation in the early stages of the hCGβ folding, a 28‐residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3‐β hairpin fragment (residues 60–87) of the hCGβ subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H₂O mixed solvents induced helical formation. Formation of β‐structure in this peptide, which may be related to the native β‐hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGβ subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGβ, is needed to induce the H3‐β hairpin formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 413–423, 1999     

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero^® anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.     

Production and scale‐up of a monoclonal antibody against 17‐hydroxyprogesterone

The hybridoma 192 was used to produce a monoclonal antibody (MAb) against 17‐hydroxyprogesterone (17‐OHP), for possible use in screening for congenital adrenal hyperplasia (CAH). The factors influencing the MAb production were screened and optimized in a 2 L stirred bioreactor. The production was then scaled up to a 20 L bioreactor. All of the screened factors (aeration rate, stirring speed, dissolved oxygen concentration, pH, and temperature) were found to significantly affect production. Optimization using the response surface methodology identified the following optimal production conditions: 36.8°C, pH 7.4, stirring speed of 100 rpm, 30% dissolved oxygen concentration, and an aeration rate of 0.09 vvm. Under these conditions, the maximum viable cell density achieved was 1.34 ± 0.21 × 10⁶ cells mL^?1 and the specific growth rate was 0.036 ± 0.004 h^?1. The maximum MAb titer was 11.94 ± 4.81 μg mL^?1 with an average specific MAb production rate of 0.273 ± 0.135 pg cell^?1 h^?1. A constant impeller tip speed criterion was used for the scale‐up. The specific growth rate (0.040 h^?1) and the maximum viable cell density (1.89 × 10⁶ cells mL^?1) at the larger scale were better than the values achieved at the small scale, but the MAb titer in the 20 L bioreactor was 18% lower than in the smaller bioreactor. A change in the culture environment from the static conditions of a T‐flask to the stirred bioreactor culture did not affect the specificity of the MAb toward its antigen (17‐OHP) and did not compromise the structural integrity of the MAb. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Crystal structure of Saccharomyces cerevisiae Aro8, a putative α‐aminoadipate aminotransferase

α‐Aminoadipate aminotransferase (AAA‐AT) catalyzes the amination of 2‐oxoadipate to α‐aminoadipate in the fourth step of the α‐aminoadipate pathway of lysine biosynthesis in fungi. The aromatic aminotransferase Aro8 has recently been identified as an AAA‐AT in Saccharomyces cerevisiae. This enzyme displays broad substrate selectivity, utilizing several amino acids and 2‐oxo acids as substrates. Here we report the 1.91Å resolution crystal structure of Aro8 and compare it to AAA‐AT LysN from Thermus thermophilus and human kynurenine aminotransferase II. Inspection of the active site of Aro8 reveals asymmetric cofactor binding with lysine‐pyridoxal‐5‐phosphate bound within the active site of one subunit in the Aro8 homodimer and pyridoxamine phosphate and a HEPES molecule bound to the other subunit. The HEPES buffer molecule binds within the substrate‐binding site of Aro8, yielding insights into the mechanism by which it recognizes multiple substrates and how this recognition differs from other AAA‐AT/kynurenine aminotransferases.     

Identification of CRISP2 from human sperm as PSP94‐binding protein and generation of CRISP2‐specific anti‐peptide antibodies

Cysteine‐rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94–CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti‐CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti‐CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Efficient synthesis of polypeptide‐α‐thioester by the method combining polypeptide expression and chemical activation for the semi‐synthesis of interferon‐γ having oligosaccharides

In order to synthesize interferon‐γ glycoform having an oligosaccharide at the 97 position by a semi‐synthetic method, interferon‐γ‐polypeptide‐(1–94)‐α‐hydrazide was prepared by the specific Cys‐cyanylation of polypeptide‐(1–94)‐Cys‐His₆ expressed from E. coli and subsequent hydrazinolysis in 22% yield (two steps). This polypeptide‐α‐hydrazide was then converted into corresponding polypeptide‐α‐thioester under NaNO₂/acid conditions followed by thiolysis in 83% yield. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

α‐Crystallin is a multimeric eye lens protein having molecular chaperone‐like function which is crucial for lens transparency. The stability and unfolding‐refolding properties of α‐crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native‐like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α‐crystallin revealed dry native‐like core of α‐crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol^?1 on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol^?1 on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α‐crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 549–560, 2014.     

Three‐dimensional structure,binding, and spectroscopic characteristics of the monoclonal antibody 43.1 directed to the carboxyphenyl moiety of fluorescein

Unlike other known anti‐fluorescein antibodies, the monoclonal antibody 43.1 is directed toward the fluorescein's carboxyl phenyl moiety. It demonstrates a very high affinity (K_D ～ 70 pM) and a fast association rate (k_on ～ 2 × 10⁷ M^?1 s^?1). The three‐dimensional structure of the Fab 43.1—fluorescein complex was resolved at 2.4 Å resolution. The antibody binding site is exclusively assembled by the CDR loops. It is comprised of a 14 Å groove‐shaped entrance leading to a 9 Å by 7 Å binding pocket. The highly polar binding pocket complementary encloses the fluorescein's carboxyphenyl moiety and tightly fixes it by multiple hydrogen bonds. The fluorescein's xanthene ring is embedded in the more hydrophobic groove and stacked between the side chains of Tyr37_L and of Arg99_H providing conditions for an excited state electron transfer process. In comparison to fluorescein, the absorption spectrum of the complex in the visible region is shifted to the “red” by 23 nm. The complex demonstrates a very weak fluorescence (Φ_c = 0.0018) with two short lifetime components: 0.03 ns (47%) and 0.8 ns (24%), which reflects a 99.8% fluorescein emission quenching effect upon complex formation. The antibody 43.1 binds fluorescein with remarkable affinity, fast association rate, and strongly quenches its emission. Therefore, it may present a practical interest in applications such as molecular sensors and switches. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 234–243, 2016.     

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

Human age‐onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens γD‐crystallin protein and its isolated domains. A partially unfolded folding intermediate of γD‐crystallin is detected in simulations with its C‐terminal domain (C‐td) folded and N‐terminal domain (N‐td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N‐td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C‐td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non‐native surface salt‐bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non‐native Glu135‐Arg142 salt‐bridge causes significant destabilization to the folding core of the isolated C‐td, which, in turn, induces unfolding of the N‐td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD‐crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD‐crystallin surface might lead to unfolding and subsequent aggregation.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

Nanostructures from the self‐assembly of α‐helical peptide amphiphiles

Self‐assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO‐(IEEYTKK)_n‐NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α‐helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α‐helical conformations under the tested conditions. The morphology of the self‐assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self‐assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self‐assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.