Valine Residue Research Articles

No association of the SUMO4 polymorphism M55V variant in type 2 diabetes in Iranian subjects

Introduction Diabetes mellitus incidence has an increasing rate and it's genetic aspect is an important approach as a risk factor and predictive value in this disorder. In some population, SUMO4, a regulator of NF-κB, gene polymorphism is associated with diabetes. A single-nucleotide polymorphism was detected in SUMO4; substituting a highly conserved methionine with a valine residue (M55V). We studied the association between M55V polymorphism in the SUMO4 gene insusceptibility of type 2 diabetes in patients with type 2 diabetes. Materials and methods Participants were 50 patients with type 2 diabetes and 50 control Iranian subjects. Genotyping was done using polymorphism chain reaction (PCR) technique and subsequent cleavage by restriction endonuclease (RFLP) for the M55V SUMO4 gene variant. Results The frequency of SUMO4 AA, AG and GG were 13%, 25% and 12% in control group and 20%, 22%, 18% in the type 2 diabetes patients respectively. The SUMO4 M55V variant was not associated with the susceptibility of type 2 diabetes. Conclusion The study indicates that the SUMO4 gene M55V variant was not associated with the susceptibility of the type 2 diabetes polymorphism.

Driving Forces for Adsorption of Amphiphilic Peptides to the Air−Water Interface

We have studied the partitioning of amphiphilic peptides at the air-water interface. The free energy of adsorption from bulk to interface was calculated by determining the potential of mean force via atomistic molecular dynamics simulations. To this end a method is introduced to restrain or constrain the center of mass of a group of molecules in a periodic system. The model amphiphilic peptides are composed of alternating valine and asparagine residues. The decomposition of the free energy difference between the bulk and interface is studied for different peptide block lengths. Our analysis revealed that for short amphiphilic peptides the surface driving force dominantly stems from the dehydration of hydrophobic side chains. The only opposing force is associated with the loss of orientational freedom of the peptide at the interface. For the peptides studied, the free energy difference scales linearly with the size of the molecule, since the peptides mainly adopt extended conformations both in bulk and at the interface. The free energy difference depends strongly on the water model, which can be rationalized through the hydration thermodynamics of hydrophobic solutes. Finally, we measured the reduction of the surface tension associated with complete coverage of the interface with peptides.

A Chinese girl molecularly diagnosed with Alagille syndrome

Alagille syndrome (AGS) is a rare or fatal disease affecting multiple systems including the liver, heart, eyes, skeleton and face. It has been considered a genetically heterogeneous disorder of the Notch signaling pathway. A 28-month-old Chinese girl with congenital heart disease and jaundice was diagnosed with Alagille syndrome by liver biopsy showing a paucity of the intrahepatic bile ducts. Variants of the JAG1 gene were detected by DNA sequencing in the patient and her unaffected father. A heterozygous missense mutation was identified in exon 2 of the JAG1 gene in the proband but not in exon 2, 4, 6, 9, 17, 23, 24 by DNA sequencing in her father. The mutation G-->T change was seen at position 133 in the cDNA sequence (c.133 G-->T), causing a substitution of a leucine for a valine (V45L) residue in the N terminus between signal peptide and DSL domain of the Notch ligand. This mutation, however, was absent in her father. Genes in the Notch signaling pathway should be further studied in AGS, and used to confirm clinical or prenatal diagnosis and facilitate genetic counseling.

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.

In Vivo Composition of NMDA Receptor Signaling Complexes Differs between Membrane Subdomains and Is Modulated by PSD-95 And PSD-93

Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids involved in the compartmentalization of signaling pathways, trafficking and sorting of proteins. At synapses, the glutamatergic NMDA receptor and its cytoplasmic scaffold protein PSD-95 move between postsynaptic density (PSD) and rafts following learning or ischemia. However it is not known whether the signaling complexes formed by these proteins are different in rafts nor the molecular mechanisms that govern their localization. To examine these issues in vivo we used mice carrying genetically encoded tags for purification of protein complexes and specific mutations in NMDA receptors, PSD-95 and other postsynaptic scaffold proteins. Isolation of PSD-95 complexes from mice carrying tandem affinity purification tags showed differential composition in lipid rafts, postsynaptic density and detergent-soluble fractions. Raft PSD-95 complexes showed less CaMKIIalpha and SynGAP and enrichment in Src and Arc/Arg3.1 compared with PSD complexes. Mice carrying knock-outs of PSD-95 or PSD-93 show a key role for PSD-95 in localizing NR2A-containing NMDA receptor complexes to rafts. Deletion of the NR2A C terminus or the C-terminal valine residue of NR2B, which prevents all PDZ interactions, reduced the NR1 association with rafts. Interestingly, the deletion of the NR2B valine residue increased the total amount of lipid rafts. These data show critical roles for scaffold proteins and their interactions with NMDA receptor subunits in organizing the differential expression in rafts and postsynaptic densities of synaptic signaling complexes.

ChemInform Abstract: A Conformationally Restricted β-Strand HIV Protease Inhibitor

The use of cyclopropanes as a conformationally restricted subunits in biological systems has been the subject of intense study by our group and others (1-10). Our recent efforts have focused on the use of 1, 2, 3-trisubstituted cyclopropanes as novel [-NH-Cα-] or [-CO-Cα-] bond replacements in pseudopeptides to restrict both side-chain orientation and enforce backbone secondary structures. To test these assumptions, the cyclopropane containing analog 1 (Fig. 1) was modeled after the potent HIV protease inhibitor 2, which together with a series of related derivatives was developed at Abbott Laboratories (11). This pseudopeptide contains a symmetrical diamino diol motif 8 (Fig. 2) flanked by Cbz-protected valine residues and is known to bind in a β-strand fashion at the enzyme-active site (12). Our analog 1 was designed to restrict the orientation of the valine residues and to mimic this extended backbone conformation. Comparison of enzyme inhibition constants for both compound 1 and the parent inhibitor 2 will then elucidate the efficacy of the cyclopropane as a conformationally restrictive subunit. Fig. 1. HIV protease inhibitors. Fig. 2. Synthesis of the cyclopropane containing inhibitor 1.

Arabidopsis Stromal-derived Factor2 (SDF2) Is a Crucial Target of the Unfolded Protein Response in the Endoplasmic Reticulum

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.

Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studies.

Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor alpha-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D(2)O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 A from a crystal of dimensions 1.60 x 0.38 x 0.38 mm corresponding to a volume of 0.23 mm(3).

Cardiac Titin

Titin constitutes the third myofilament of cardiac muscle, with a single giant polypeptide spanning from Z-disk to the M-band region of the sarcomere1 (Fig. 1). The ∼1.0 MDa region in the I-band is extensible and consists of tandemly arranged immunoglobulin (Ig)-like domains that make up proximal (near Z-disk) and distal (near A-I junction) segments, interspersed by the PEVK sequence (rich in proline, glutamate, valine, and lysine residues) and the N2B element2. Each functions as a distinct spring element3. The C-terminal ∼2 MDa of titin is located in the A-band and is inextensible. It is composed of regular arrays of Ig and fibronectin type 3 (Fn3) modules forming so-called super-repeats2. A-band titin may function as a molecular ruler, regulating assembly of the thick filament2,4,5. Titin's ∼250-kDa COOH-terminal region is an integral part of the M-band and contains a kinase domain6,7. As in the Z-disk, where titin filaments from opposite sarcomeres overlap, titin filaments from opposite half-sarcomeres overlap within the M-band, where they are interconnected by M-band proteins8. Thus, titin filaments with opposite polarity overlap in both Z-disk and M-band, forming a contiguous filament along the myofibril. In this review, we discuss titin's functions in the heart, with an emphasis on its role in diastolic function and the various mechanisms whereby passive stiffness can be tuned. Due to space constraints, it has not been possible to provide inclusive references to all original articles in the field. Figure 1 Schematic of titin in sarcomere. Differential splicing Titin is encoded by a single gene containing 368 exons. Multiple splice pathways in the I-band encoding region (∼230 exons) give rise to isoforms with different spring composition9. The three cardiac isoform classes are shown in Fig. 1. The relatively small ∼3.0 MDa isoform is known as N2B titin (it contains the N2B element)9. A second class also contains the N2A element, and is termed N2BA titin. N2BA titins have a longer PEVK segment and a variable number of additional Ig domains resulting in a ∼3.3-3.5 MDa size9. The third class includes isoforms that predominate in fetal-neonatal life which contain additional spring elements in both tandem Ig and PEVK regions, resulting in a ∼3.6-3.8 MDa size protein10-12. These isoforms gradually disappear during postnatal development. Regulation of the spring composition of titin in fetal-neonatal myocardium allows adjustment of diastolic filling properties during development.

Screening of novel dominant negative mutant actins using glycine targeted scanning identifies G146V actin that cooperatively inhibits cofilin binding

A number of studies suggested that the structure of actin filaments changes by interaction with actin-binding proteins such as cofilin and myosin, and that the conformational changes of the actin subunits within a filament are cooperative. To understand the functions of these cooperative conformational changes induced by actin-binding proteins, we sought to obtain dominant negative mutant actins impaired in cooperative conformational changes. A series of mutant actin genes in which glycine residues in actin were systematically substituted by valine residues were constructed, and were expressed individually in yeast cells that carry a wild-type endogenous actin gene. Six dominant negative actin mutations were identified on the basis of growth inhibition. Among them, G146V mutation was chosen for further biochemical analysis because the Gly146 residue is located at the strategic hinge position connecting the large and small domains of an actin molecule. We found that G146V actin filaments hardly bind cofilin, consistent with a previous suggestion that cofilin binding causes conformational changes of actin around Gly146 (Galkin et al. [3]). Notably, copolymer that consists of 1:10 mixture of the mutant and wild-type actin molecules showed significantly reduced affinity for cofilin, suggesting that G146V mutant actin affects the conformation of neighboring wild-type actin within a filament, and inhibits cofilin binding.

Biochemical and Structural Characterization of Two Site-Directed Mutants of Staphylococcus xylosus Lipase

Staphylococcus xylosus AF208229 lipase was expressed in E. coli containing an histidine-tag (WT-Val). In the present work, in order to check the importance of the residue 309 in the specific activity, the amino acid side chain residue valine 309 was substituted by aspartate or lysine through site-directed mutagenesis. Both mutant lipases (MUT-Lys and MUT-Asp) were expressed in E. coli and the recombinant histidine-tagged lipases were purified by immobilized metal ion affinity chromatography. The enzyme activity was determined using p-nitrophenyl butyrate as substrate and secondary structure content was evaluated by circular dichroism. MUT-Lys and MUT-Asp presented significant increase of lipase activity (P < 0.05) in comparison to WT-Val, although highest activities for the three enzymes were observed at the same pH and temperature (pH 9.0 and 42 degrees C). The wild type and mutant lipases presented high thermal stability, after 30 min of incubation at 80 degrees C all enzymes retained their initial activities.

Enhancing the catalytic activity of an engineered threonine protease

In this study, the Cys‐42—Cys‐58 disulfide bridge of S195T trypsin was replaced with two valine residues to: 1) increase threonine protease activity and 2) better understand the structual limitations and requirements of this subsite with respect to catalysis. The activities of C42V/C58V and C42V/C58V/S195T trypsins (VV‐Tn and VVT‐Tn, respectively) were evaluated with benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) p‐nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) substrates. VVT‐Tn was 150 and 10 times more active towards the pNA and AMC substrates, respectively, than C42A/C58V/S195T trypsin (AVT‐Tn; kcat/KM values). In a thermostability study, both variants and WT trypsin retained &gt; 80% of their activity suggesting that the substitutions did not destabilize the enzymes. The pH dependence of activity profiles were identical to WT trypsin, suggesting that any structural differences do not perturb the pKas of catalytically relevant residues. Finally, the KI of p‐aminobenzamidine was identical for native and VV‐Tn but that of VVT‐Tn was half the KI for WT. Therefore, Thr‐195 may affect the primary binding pocket structure. Collectively, these results indicate that the activity of Thr‐195 trypsin variants may be increased by appropriate substitutions at positions 42 and 58. However, the structural pertubations induced by these substitutions make achievement of wild‐type levels of activity less likely.(This work was supported by NSF Award MCB‐0643988‐02)

Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High‐Molecular‐Weight Proteins

Sometimes less is more: [13C1H3]methyl isotopomers can be biosynthetically incorporated specifically into the pro-S methyl groups of leucine and valine residues in large protein assemblies within a perdeuterated background by using an acetolactate precursor. This stereospecific labeling strategy considerably enhances NMR spectra for large protein assemblies. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Comprehensive and Cost-Effective NMR Spectroscopy of Methyl Groups in Large Proteins

An NMR approach is described which yields the methyl resonance assignments of alanine, threonine, valine, leucine, and isoleucine residues in proteins with high sensitivity and excellent resolution. The method relies on protein samples produced by bacterial expression using [(1)H,(13)C]-D-glucose and approximately 100% D(2)O, which is cost-effective and ensures the isotopic enrichment of all possible methyl groups. Magnetization transfer throughout the methyl-containing side chains is possible with this labeling scheme due to the high level of deuteration along the amino acid side chain, coupled with the selection of the favorable CHD(2) methyl isotopomer for detection. In an application to the 34 kDa periplasmic binding protein FepB 164 out of 195 methyl groups (85%) were assigned sequence-specifically and stereospecifically. This percentage increases to 91% when taking into account that not all backbone assignments are available for this system. The remaining unassigned methyl groups belong to six leucine residues, caused by low cross-peak intensities, and four alanine residues due to degeneracy of the (13)C(alpha)/(13)C(beta) frequencies. Our results demonstrate that NMR spectroscopic investigations of protein structure, dynamics, and interactions can be extended to include all methyl-containing amino acids also for larger proteins.

An Arabidopsis Hydrophilic Ca2+-Binding Protein with a PEVK-Rich Domain, PCaP2, is Associated with the Plasma Membrane and Interacts with Calmodulin and Phosphatidylinositol Phosphates

We found a new hydrophilic protein in Arabidopsis thaliana. Real-time PCR demonstrated that the protein was expressed in roots. Histochemical analysis of promoter-beta-glucuronidase fusions demonstrated its extensive expression in root hairs. The protein is rich in proline, glutamate, valine and lysine residues (PEVK-rich domain), and bound Ca(2+) even in the presence of Mg(2+) and K(+) when examined by the (45)Ca overlay assay. Treatment of seedlings with K(+), Mn(2+), Zn(2+), Na(+), ABA and gibberellic acid, and cold and drought stresses enhanced the transcription. Expression of the protein linked to green fluorescent protein in A. thaliana showed its plasma membrane localization and cell-specific expression in the epidermal cells including root hairs and the elongating pollen tubes. Therefore, we named the protein PCaP2 (plasma membrane-associated Ca(2+)-binding protein-2). The substitution of glycine at position 2 with alanine resulted in cytoplasmic localization of PCaP2. These results and the N-terminal characteristic motif suggest that PCaP2 is N-myristoylated at Gly2. We examined the capacity for binding to phosphatidylinositol phosphates (PtdInsPs), and found that PCaP2 interacts strongly with PtdIns(3,5)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), and weakly with PtdIns(3,4)P(2). Furthermore, calmodulin was associated with PCaP2 in a Ca(2+)-dependent manner, and its association weakened the interaction of PCaP2 with PtdInsPs. These results indicate that PCaP2 is involved in intracellular signaling through interaction with PtdInsPs and calmodulin in growing root hairs. PCaP2 was previously reported as microtubule-associated protein-18. We discuss the physiological roles of PCaP2 in relation to microtubules in cells.

Intrinsic Propensities of Amino Acid Residues in GxG Peptides Inferred from Amide I′ Band Profiles and NMR Scalar Coupling Constants

A reliable intrinsic propensity scale of amino acid residues is indispensable for an assessment of how local conformational distributions in the unfolded state can affect the folding of peptides and proteins. Short host-guest peptides, such as GxG tripeptides, are suitable tools for probing such propensities. To explore the conformational distributions sampled by the central amino acid residue in these motifs, we combined vibrational (IR, Raman, and VCD) with NMR spectroscopy. The data were analyzed in terms of a superposition of two-dimensional Gaussian distribution functions in the Ramachandran space pertaining to subensembles of polyproline II, beta-strand, right- and left-handed helical, and gamma-turn-like conformations. The intrinsic propensities of eight amino acid residues (x = A, V, F, L, S, E, K, and M) in GxG peptides were determined as mole fractions of these subensembles. Our results show that alanine adopts primarily (approximately 80%) a PPII-like conformation, while valine and phenylalanine were found to sample PPII and beta-strand-like conformations equally. The centers of the respective beta-strand distributions generally do not coincide with canonical values of dihedral angles of residues in parallel or antiparallel beta-strands. In fact, the distributions for most residues found in the beta-region significantly overlap the PPII-region. A comparison with earlier reported results for trivaline reveals that the terminal valines increase the beta-strand propensity of the central valine residue even further. Of the remaining investigated amino acids, methionine preferred PPII the most (0.64), and E, S, L, and K exhibit moderate (0.56-0.45) PPII propensities. Residues V, F, S, E, and L sample, to a significant extent, a region between the canonical PPII and (antiparallel) beta-strand conformations. This region coincides with the sampling reported for L and V using theoretical predictions (Tran et al. Biochemistry 2005, 44, 11369). The distributions of all investigated residues differ from coil library and computationally predicted distributions in that they do not exhibit a substantial sampling of helical conformations. We conclude that this sampling of helical conformations arises from the context dependence, for example, neighboring residues, in proteins and longer peptides, some of which is long-range.

The importance of valine 114 in ligand binding in β2‐adrenergic receptor

G-protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. beta(2)-Adrenergic receptor (beta(2)-AR) is a well-studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand-binding region of beta(2)-AR using the recently solved high-resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure-function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster beta(2)-AR by site-directed mutagenesis. We replaced V114 in hamster beta(2)-AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G-protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side-chains but also the aryl-ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in beta(2)-AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding.

GRASP65 and GRASP55 Sequentially Promote the Transport of C-terminal Valine-bearing Cargos to and through the Golgi Complex

The Golgi matrix proteins GRASP65 and GRASP55 have recognized roles in maintaining the architecture of the Golgi complex, in mitotic progression and in unconventional protein secretion whereas, surprisingly, they have been shown to be dispensable for the transport of commonly used reporter cargo proteins along the secretory pathway. However, it is becoming increasingly clear that many trafficking machineries operate in a cargo-specific manner, thus we have investigated whether GRASPs may control the trafficking of selected classes of cargo. We have taken into consideration the C-terminal valine-bearing receptors CD8alpha and Frizzled4 that we show bind directly to the PSD95-DlgA-zo-1 (PDZ) domains of GRASP65 and GRASP55. We demonstrate that both GRASPs are needed sequentially for the efficient transport to and through the Golgi complex of these receptors, thus highlighting a novel role for the GRASPs in membrane trafficking. Our results open new perspectives for our understanding of the regulation of surface expression of a class of membrane proteins, and suggests the causal mechanisms of a dominant form of autosomal human familial exudative vitreoretinopathy that arises from the Frizzled4 mutation involving its C-terminal valine.

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe(III)NO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe(III)NO dissociation rate (k(d)) and NO association rate (k(on)) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k(on) and k(d) by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O(2) catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O(2) intermediate, but instead were due to the slower NO k(on) and k(d) and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.

Sequence-dependent gelation kinetics of β-hairpin peptide hydrogels.

The gelation kinetics of four β-hairpin oligopeptides that have been designed to exhibit responsive behavior to changes in environmental conditions, such as pH, ionic strength and temperature, are characterized using multiple particle tracking microrheology and circular dichroism (CD) spectroscopy. The peptides, predominantly an alternating sequence of valine and lysine residues, differ by a point substitution of a single amino acid near a type II'β-turn sequence. The rate of gelation becomes faster for point substitutions which reduce the total charge of the peptide. Similarly, increasing the ionic strength reduces or screens intra- and inter-molecular electrostatic repulsions, again leading to faster gelation kinetics. CD measurements show that the concentration of folded peptide at the gel point decreases as the gelation kinetics become slower, possibly indicating a relationship between the assembly rate and the resulting gel microstructure. Finally, a model is developed based on the electrostatic barrier to peptide folding and association which agrees semi-quantitatively with the microrheology results. This represents a first step towards understanding the role of peptide charge and physico-chemical conditions in the self-assembly of these peptide hydrogelators.