Gly Replacement Research Articles

Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa)n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502GFPGER507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI.

NMR Conformational and Dynamic Consequences of a Gly to Ser Substitution in an Osteogenesis Imperfecta Collagen Model Peptide

Close packing of three chains in a standard collagen triple helix requires Gly as every third residue. Missense mutations replacing one Gly by a larger residue in the tripeptide repeating sequence in type I collagen are common molecular causes of osteogenesis imperfecta. The structural and dynamic consequences of such mutations are addressed here by NMR studies on a peptide with a Gly-to-Ser substitution within an alpha1(I) sequence. Distances derived from nuclear Overhauser effects indicate that the three Ser residues are still packed in the center of the triple helix and that the standard 1-residue stagger is maintained. NMR dynamics using H-exchange and temperature-dependent amide chemical shifts indicate a greater disruption of hydrogen bonding and/or increased conformational flexibility C-terminal to the Ser site when compared with N terminal. This is consistent with recent suggestions relating clinical severity with an asymmetric effect of residues N- versus C-terminal to a mutation site. Dynamic studies also indicate that the relative position between a Gly in one chain and the mutation site in a neighboring staggered chain influences the disruption of the standard hydrogen-bonding pattern. The structural and dynamic alterations reported here may play a role in the etiology of osteogenesis imperfecta by affecting collagen secretion or interactions with other matrix molecules.

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in l-galactono-gamma-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or a Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala-113 --> Gly) resulted in a striking 400-fold increase in oxygen reactivity, whereas the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (k(ox) = 3.5 x 10(5) m(-1) s(-1)), similar to most other flavoprotein oxidases. The Ala-113 --> Gly replacement does not change the reduction potential of the flavin but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme, Ala-113 acts as a gatekeeper, preventing oxygen from accessing the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family and is absent in members that act as oxidases.

Critical Amino Acid Residues for Nicotine 5′-Hydroxylation in Human CYP2A Enzymes

Objective We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-(3-pyridyl)-1-butanone(NNK) and aflatoxin B1(AFB1) carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods A series of reciprocally substituted mutants of CYP2A6Ile 300 → Phe, CYP2A6Gly 301Ala, CYP2A6Ser 369 → Gly, CYP2A13Phe 300 → Ile, CYP2A13Ala 301 → Gly and CYP2A13Gly 369 → Ser were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5′hydroxylatin by wild type and mutant CYP2A proteins was performed. Results All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile 300 → Phe and CYP2A6Gly 301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe 300→Ile and CYP2A13Ala 301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by V max variations rather than K m changes. Substitution of #369 residue significantly affected both K m and V max values. CYP2A6Ser 369 → Gly increase the catalytic efficiency via a significant K m decrease versus V max enhancement, while the opposite effects were seen with CYP2A13Gly 369 → Ser. Conclusion #300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5′-oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Ser 369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu 370 which is crucial for many hydroxylations.

Clearance mechanisms of von Willebrand factor and factor VIII.

Clearance mechanisms of von Willebrand factor and factor VIII.

Rigidification of a Flexible Protease Inhibitor Variant upon Binding to Trypsin

The Tyr35 → Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 Å resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 Å data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.

Stability Junction at a Common Mutation Site in the Collagenous Domain of the Mannose Binding Lectin

Missense mutations in the collagen triple-helix that replace one of the required Gly residues in the (Gly-Xaa-Yaa)(n)() repeating sequence have been implicated in various disorders. Although most hereditary collagen disorders are rare, a common occurrence of a Gly replacement mutation is found in the collagenous domain of mannose binding lectin (MBL). A Gly --> Asp mutation at position 54 in MBL is found at a frequency as high as 30% in certain populations and leads to increased susceptibility to infections. The structural and energetic consequences of this mutation are investigated by comparing a triple-helical peptide containing the N-terminal Gly-X-Y units of MBL with the homologous peptide containing the Gly to Asp replacement. The mutation leads to a loss of triple-helix content but only a small decrease in the stability of the triple-helix (DeltaT(m) approximately 2 degrees C) and no change in the calorimetric enthalpy. NMR studies on specifically labeled residues indicate the portion of the peptide C-terminal to residue 54 is in a highly ordered triple-helix in both peptides, while residues N-terminal to the mutation site have a weak triple-helical signal in the parent peptide and are completely disordered in the mutant peptide. These results suggest that the N-terminal triplet residues are contributing little to the stability of this peptide, a hypothesis confirmed by the stability and enthalpy of shorter peptides containing only the region C-terminal to the mutation site. The Gly to Asp replacement at position 54 in MBL occurs at the boundary of a highly stable triple-helix region and a very unstable sequence. The junctional position of this mutation minimizes its destabilizing effect, in contrast with the significant destabilization seen for Gly replacements in peptides modeling collagen diseases.

Three-Dimensional Model and Molecular Mechanism ofMycobacterium tuberculosisCatalase-Peroxidase (KatG) and Isoniazid-Resistant KatG Mutants

Mycobacterium tuberculosis KatG enzyme functions both as catalase for removing hydrogen peroxide (H(2)O(2)) and as peroxidase for oxidating isoniazid (INH) to active form of anti-tuberculosis drug. Although mutations in M. tuberculosis KatG confer INH resistance in tuberculous patients, structural bases for INH-resistant mutations in the KatG gene remains poorly understood. Here, three M. tuberculosis KatG mutants bearing Arg418--> Gln, Ser315 --> Thr, or Trp321 --> Gly replacement were assessed for changes in catalase-peroxidase activities and possible structure bases relevant to such changes. These three M. tuberculosis KatG mutants exhibited a marked impairment or loss of catalase-peroxidase activities. The possible structural bases for the mutant-induced loss of enzyme activities were then analyzed using a three-dimensional model of M. tuberculosis KatG protein constructed on the basis of the crystal structure of the catalase-peroxidase from Burkholderia pseudomallei. The model suggests that three M. tuberculosis KatG mutants bearing Arg418 --> Gln, Ser315 -->Thr, or Trp321--> Gly replacement affect enzyme activities by different mechanisms, although each of them impacts consequently on a heme-associated structure, the putative oxidative site. Moreover, in addition to the widely accepted substrate-binding site, M. tuberculosis KatG may bear another H(2)O(2) binding site. This H(2)O(2) binding site appears to interact with the catalytic site by a possible electron-transfer chain, a Met255-Tyr229-Trp107 triad conserved in many catalase-peroxidases. The Ser315 --> Thr mutant may have direct effect on the catalytic site by interfering with electron transfer in addition to the previously proposed mechanism of steric constraint.

Stability related bias in residues replacing glycines within the collagen triple helix (Gly-Xaa-Yaa) in inherited connective tissue disorders.

A missense mutation leading to the replacement of one Gly in the (Gly-Xaa-Yaa)n repeat of the collagen triple helix can cause a range of heritable connective tissue disorders that depend on the gene in which the mutation occurs. Osteogenesis imperfecta results from mutations in type I collagen, Ehlers-Danlos syndrome type IV from mutations in type III collagen, Alport syndrome from mutations in type IV collagen, and dystrophic epidermolysis bullosa from mutations in type VII collagen. The predicted rates of substitutions by different amino acids for glycine in the alpha1(I), alpha2(I), alpha1(III), alpha5(IV), and alpha1(VII) chains (encoded by COL1A1, COL1A2, COL3A1, COL4A5, and COL7A1, respectively) were compared with missense mutations in those chains that have been observed to cause disease. The spectrum of amino acids replacing Gly was not significantly different from that expected for the alpha1(VII) chains, suggesting that any Gly replacement will cause disease. The distribution of residues replacing Gly was significantly different from that expected for all other collagen chains studied, with a particularly strong bias seen for alpha1(I) and alpha1(III) collagen chains. The bias did not correlate with the degree of chemical dissimilarity between Gly and the replacement residues, but in some cases a relationship was observed with the predicted extent of destabilization of the triple helix. For alpha1(III) collagen chains, the more destabilizing mutations were identified more often than expected. For alpha1(I), the most destabilizing residues, Val, Glu, and Asp, and the least destabilizing residue, Ala, were underrepresented. This bias supports the hypothesis that the level of triple-helix destabilization determines clinical outcome.

The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and 15N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His → Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS∗L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)3 donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS∗N donor set deriving from His46, Cys112 and Met121. The Cu-Sδ(Met) distance is reduced from about 3.2 to 2.3 Å. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the β-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.

Design of salt-insensitive glycine-rich antimicrobial peptides with cyclic tricystine structures.

Cyclic peptide backbone and cystine constraints were used to develop a broadly active salt-insensitive antimicrobial peptide [Gly(6)]ccTP 1a with eight Gly residues in an 18-residue sequence. The importance of rigidity and amphipathicity imparted by the cyclic and cystine constraints was examined in two peptide series based on tachyplesin, a known beta-stranded antimicrobial peptide. The first series, which retained the charge and hydrophobic amino acids of tachyplesin, but contained zero to four covalent constraints, included a cyclic tricystine tachyplesin (ccTP 1). Corresponding [Gly(6)] analogues were prepared in a parallel series with all six bulky hydrophobic amino acids in their sequences replaced with Gly. Circular dichroism measurements showed that ccTP 1 and [Gly(6)]ccTP 1a exhibited well-ordered beta-sheet structures, while the less constrained [Gly(6)] analogues were disordered. Except for linear peptides assayed under high-salt conditions, peptides with increased or decreased conformational constraints retained broad activity spectra with small variations in potency of 2-10-fold compared to that of tachyplesin. In contrast, Gly replacement analogues resulted in large variations in activity spectra and significant decreases in potency that roughly correlated with the decreases in conformational constraints. Except against Escherichia coli, the Gly-rich analogues with two or fewer covalent constraints were largely inactive under high-salt conditions. Remarkably, the most constrained [Gly(6)]ccTP 1a retained a broad activity spectrum against all 10 test microbes in both low- and high-salt assays. Collectively, our results show that [Gly(6)]ccTP 1acould serve as a template for further analogue study to improve potency and specificity through single or multiple replacements of hydrophobic or unnatural amino acids.

Carboxin resistance in Paracoccus denitrificans conferred by a mutation in the membrane-anchor domain of succinate:quinone reductase.

Succinate:quinone reductase is a membrane-bound enzyme of the citric acid cycle and the respiratory chain. Carboxin is a potent inhibitor of the enzyme of certain organisms. The bacterium Paracoccus denitrificans was found to be sensitive to carboxin in vivo, and mutants that grow in the presence of 3'-methyl carboxin were isolated. Membranes of the mutants showed resistant succinate:quinone reductase activity. The mutation conferring carboxin resistance was identified in four mutants. They contained the same missense mutation in the sdhD gene, which encodes one of two membrane-intrinsic polypeptides of the succinate:quinone reductase complex. The mutation causes an Asp to Gly replacement at position 89 in the SdhD polypeptide. P. denitrificans strains that overproduced wild-type or mutant enzymes were constructed. Enzymic properties of the purified enzymes were analyzed. The apparent Km for quinone (DPB) and the sensitivity to thenoyltrifluoroacetone was normal for the carboxin-resistant enzyme, but the succinate:quinone reductase activity was lower than for the wild-type enzyme. Mutations conferring carboxin resistance indicate the region on the enzyme where the inhibitor binds. A previously reported His to Leu replacement close to the [3Fe-4S] cluster in the iron-sulfur protein of Ustilago maydis succinate:quinone reductase confers resistance to carboxin and thenoyltrifluoroacetone. The Asp to Gly replacement in the P. denitrificans SdhD polypeptide, identified in this study to confer resistance to carboxin but not to thenoyltrifluoroacetone, is in a predicted cytoplasmic loop connecting two transmembrane segments. It is likely that this loop is located in the neighborhood of the [3Fe-4S] cluster.

A New Hemoglobin Variant Found During Hb A1c Measurement: Hb Hokusetsu [β52(D3)Asp→Gly

A new β chain variant was accidentally found through the assay of Hb A1c in a diabetic patient. The variant was detected by polyacrylamide gel isoelectrofocusing and electrospray ionization mass spectrometry. For sequence determination, globin was cleaved with combination of trypsin and lysyl endopeptidase and analyzed by high performance liquid chromatography connected to electrospray ionization mass spectrometry. An abnormal βT-5 peptide was found by reconstructed selected ion monitoring. The collision-induced dissociation spectrum of an ion derived from the abnormal βT-5 peptide revealed a new substitution, [β52(D3)Asp→Gly], named Hb Hokusetsu. The sequence was confirmed with an automatic sequencer using peptides isolated by reversed phase high performance liquid chromatography. Amplification of the β-globin exon 2 and nucleotide sequencing revealed a GATG`T mutation in codon 52 corresponding to an Asp→Gly replacement. Electrospray ionization mass spectrometry analysis of the hemolysate showed a reasonable value of 10.4% for glycated globin. The variant migrated as Hb S on isoelectrofocusing. Hematological analysis revealed normal parameters. The patient's hemolysate showed normal stability in the isopropanol test. Oxygen equilibrium studies on the patient's red blood cells and hemolysate showed no significant change in oxygen affinity or cooperativity.

Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation.

The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165-->Ala, Lys166-->Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158-->Arg, Ser160-->Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TF(R158G160). In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TF(A165A166) as the cofactor, but only 3.5-fold with TF(R165G160). The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TF(R158G160) and 7-8-fold with TF(A165A166). This suggests that the molecular recognition of VII in complex with TF by the enzymes TF-VIIa and factor Xa are similar. Generation of factor IXa by TF(R158G160)-VIIa was unaltered, but reduced 2-fold with TF(A165A166). In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TF-VIIa complex.

Glycine attenuates Fanconi syndrome induced by maleate or ifosfamide in rats

It has become widely recognized that glycine (Gly) depletion predisposes isolated proximal tubules (PT) to necrotic cell damage induced by diverse insults and that Gly replacement in vitro is highly cytoprotective. However, the effectiveness of supplementation with Gly in vivo, where blood and tissue Gly normally are maintained at high levels, is incompletely defined. Our aim was to assess whether: (a) supplementation of Gly in drinking water of rats would attenuate the proximal tubule damage and the Fanconi syndrome (FS) induced by maleate (Mal), a classical proximal tubule toxin, or ifosfamide (IFO), an antineoplastic drug; and (b) to explore the mechanisms responsible for such effects, since Gly supplementation might be especially beneficial in treating the FS, where the kidney tends to waste amino acids. Rats received daily injection of Mal (2 mmol/kg) for two days without or with oral supplementation of 2% Gly. IFO, 50 mg/kg, was injected daily for five days without or with oral Gly. Control rats were injected with saline, without or with oral Gly. The results demonstrated that both Mal and IFO induced a FS characterized by wasting of amino and organic acids, glucose, and electrolytes, along with elevated plasma creatinine (Crn) and BUN, and decreased Crn clearance rate. Light microscopy revealed a necrotic lesion in the proximal tubules of the Mal group, but no necrosis after IFO. Gly strongly ameliorated the severity of renal necrosis and/or dysfunction induced by Mal or IFO, with significant decreases in total and fractional excretion of Na+, K+, PO4(3-) and glucose, decreased plasma BUN and Crn, and increased Crn clearance. Analysis of freeze-clamped cortical tissue showed substantial depletion of [Gly], [ATP] and [GSH] along with increased GSSG in Mal or IFO groups and correction of [Gly] and [ATP] with Gly supplementation, but no improvement with Gly of reduced gluthatione [GSH] or the ratio of reduced to oxidized gluthatione (GSH/GSSG). 31P-NMR analysis of the renal cortex indicated a decrease in Pi and various membrane phospholipids in Mal and IFO rats and prevention of this damage with Gly. These observations demonstrate that oral supplementation of Gly can provide protection against Mal or IFO-induced renal tubular cell dysfunction and structural damage. The lack of effect on glutathione oxidation and depletion suggests an action distal to toxin uptake and intracellular interactions, which is similar to the characteristics of Gly cytoprotection against diverse insults in vitro. The results also suggest modification by Gly of the primary toxicity of the agents and effects on phospholipid synthesis that could contribute to repair.

Development of a Novel Recombinant Serpin with Potential Antithrombotic Properties

Recombinant alpha 1-antitrypsin with a P1 arginine residue (Arg-alpha 1-antitrypsin) is a rapid inhibitor of both thrombin and activated protein C (APC). A series of mutants were made in an attempt to increase the specificity of this serpin for thrombin over APC. Initially, P2 and P'1 residues of Arg-alpha 1-antitrypsin were replaced in single and double mutations by the corresponding residues in antithrombin and C1 inhibitor which are very poor inhibitors of APC. No improvement in selectivity was achieved by these mutations. In fact, all P2/P'1 substitutions led to a decrease in selectivity for thrombin over APC. For example, replacement of the P2 proline of Arg-alpha 1-antitrypsin by glycine decreased the association rate constant (kass) with thrombin by 37-fold while the kass value with APC was reduced by only 16-fold. Cooperative effects were observed with the double P2 and P'1 substitutions; the mutational effects were not additive. The decrease in the kass for thrombin caused by the mutation of the P2 proline to alanine or glycine was 3-fold greater when threonine was present in the P'1 position instead of the normal serine. In contrast to the disappointing results with the P2/P'1 mutations, replacement of the P7 to P'3 residues of alpha 1-antitrypsin by those of antithrombin led to a dramatic increase in selectivity. Although this substitution only affected the kass value with thrombin by 10-fold, a 12,500-fold decrease in this value with APC was observed. Substitution of proline for the P2 glycine of this chimeric serpin increased the kass values with thrombin and APC by 7- and 90-fold, respectively. The effect of the P2 substitution was again found to depend on the sequence surrounding the residue; the change in the kass for APC caused by the P2 Pro-->Gly replacement was 6-fold larger in the chimeric serpin. Evaluation of the kass values of the chimeric serpin with a P2 proline in light of the likely rates of inhibition of thrombin and APC during antithrombotic therapy with heparin suggested that this serpin may have kinetic parameters suitable for an antithrombotic agent.

Drosophila GABA-gated chloride channel: modified [3H]EBOB binding site associated with Ala-->Ser or Gly mutants of Rdl subunit.

The non-competitive blocker site of the GABA-gated chloride ion channel in normal susceptible strains of Drosophila melanogaster and simulans binds 4-n-[3H]propyl-4'-ethynylbicycloorthobenzoate ([3H]EBOB) at specific sites with KdS of 1.6-1.9 nM and BmaxS of 171-181 fmol/mg protein. This specific binding of [3H]EBOB is strongly inhibited by: a large number and variety of insecticidal channel blockers at 20 nM (lindane, alpha-endosulfan, dieldrin, 12-ketoendrin, fipronil, and a representative bicycloorthobenzoate and dithiane) or 200 nM (picrotoxinin); the insecticidal channel activators avermectin and moxidectin at 20 nM; muscimol at 30 microM and GABA at 300 microM. Cyclodiene resistance in D. melanogaster has been attributed to a mutation resulting in an Ala302-->Ser replacement in the Rdl GABA receptor subunit and in D. simulans to an homologous Ala-->Ser or Gly replacement. These mutations are shown here to greatly reduce [3H]EBOB binding, i.e. lower affinity and apparent number of binding sites. The Ala-->Ser replacement with both melanogaster and simulans almost always reduces the potency in inhibiting [3H]EBOB binding of each of eight channel blockers and of muscimol and GABA. The Ala-->Gly replacement in D. simulans is generally less effective than the Ala-->Ser modification in reducing sensitivity to the channel blockers and to muscimol and GABA. The channel activators avermectin and moxidectin usually retain their inhibitory potency in the Rdl subunit mutants. Thus, it appears that replacement of Ala by Ser generally modifies the non-competitive blocker site and its coupling to the GABA-recognition site with less effect on the channel activator site. In contrast, the Ala-->Gly replacement has less impact in protecting the chloride channel from the action of insecticidal blockers. Each of the resistant strains has the same level of resistance to the lethal action of the five channel blockers examined but none to avermectins and muscimol.

Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes.

On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme.

Contributions of left-handed helical residues to the structure and stability of bacteriophage T4 lysozyme

Non-glycine residues in proteins are rarely observed to have “left-handed helical” conformations. For glycine, however, this conformation is common. To determine the contributions of left-handed helical residues to the stability of a protein, two such residues in phage T4 lysozyme, Asn55 and Lys124, were replaced with glycine. The mutant proteins fold normally and are fully active, showing that left-handed non-glycine residues, although rare, do not have an indispensable role in the folding of the protein or in its activity. The thermodynamic stability of the Lys124 to Gly variant is essentially identical with that of wild-type lysozyme. The Asn55 to Gly mutant protein is marginally less stable (0.5 kcal/ mol). These results indicate that the conformational energy of a glycine and a non-glycine residue in the left-handed helical conformation are very similar. This is consistent with some theoretical energy distributions, but is inconsistent with others, which suggest that replacements of the sort described here might increase the stability of the protein by up to 5 kcal/mol. Crystallographic analysis of the mutant proteins shows that the backbone conformation of the Lys124 to Gly variant is essentially identical with that of the wild-type structure. In the case of the Asn55 to Gly replacement, however, the (φ,ψ) values of residue 55 change by about 20 °. This suggests that the energy minimum for left-handed glycine residues is not the same as that for non-glycine residues. This is strongly indicated also by a survey of accurately determined protein crystal structures, which suggests that the energy minimum for left-handed glycine residues is near (φ = 90 °, ψ = 0 °), whereas that for non-glycine residues is close to (φ = 60 °, ψ = 30 °). This apparent energy minimum for glycine is not clearly predicted by any of the theoretical (φ,ψ) energy contour maps.