Leu Mutant Research Articles

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

Effect of Individual Fc Methionine Oxidation on FcRn Binding: Met252 Oxidation Impairs FcRn Binding More Profoundly than Met428 Oxidation

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies. However, the quantitative effect of individual Met oxidation on Fc-FcRn binding has not been addressed. This information is valuable for defining critical quality attributes. In the present study, two sets of homodimeric site-directed IgG1 mutations were generated to understand how individual Fc Met oxidation affects FcRn binding. The first approach used Met to Leu mutants to block site-specific Met oxidation. In the other approach, Met to Gln mutants were designed to mimic site-specific Met oxidation. Both mutagenesis approaches show that either Met252 or Met428 oxidation alone significantly impairs Fc-FcRn binding. Met252 oxidation has a more deleterious effect on FcRn binding than M428 oxidation, whereas Met428 oxidation has a bigger destabilization effect on the thermal stability. Our results also show that Met358 oxidation does not affect FcRn binding. In addition, our study suggests that Met to Gln mutation may serve as an important tool to understand Met oxidation.

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu(940) in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu(940) of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67-100% of (α1→6) linkages) were produced by GTF180 and its Leu(940) mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu(940) in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.

Mutagenesis and Computational Modeling of Human G-Protein-Coupled Receptor Y2 for Neuropeptide Y and Peptide YY

Neuropeptide Y and peptide YY receptor type 2 (Y2) is involved in appetite regulation and several other physiological processes. We have investigated the structure of the human Y2 receptor. Computational modeling of receptor-agonist interactions was used as a guide to design a series of receptor mutants, followed by binding assays using full-length and truncated peptide agonists and the Y2-specific antagonist BIIE0246. Our model suggested a hydrogen bond network among highly conserved residues Thr2.61, Gln3.32, and His7.39, which could play roles in ligand binding and/or receptor structure. In addition, the C-terminus of the peptide could make contact with residues Tyr5.38 and Leu6.51. Mutagenesis of all these positions, followed by binding assays, provides experimental support for our computational model: most of the mutants for the residues forming the proposed hydrogen bond network displayed reduced peptide agonist affinities as well as reduced hPYY3-36 potency in a functional assay. The Ala and Leu mutants of Gln3.32 and His7.39 disrupted membrane expression of the receptor. Combined with the modeling, the experimental results support roles for these hydrogen bond network residues in peptide binding as well as receptor architecture. The reduced agonist affinity for mutants of Tyr5.38 and Leu6.51 supports their role in a binding pocket surrounding the invariant tyrosine at position 36 of the peptide ligands. The results for antagonist BIIE0246 suggest several differences in interactions compared to those of the peptides. Our results lead to a new structural model for NPY family receptors and peptide binding.

The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.

Identification of dominant negative effect of L522P mutation in the electrogenic Na+–HCO3 − cotransporter NBCe1

Homozygous mutations in the electrogenic Na(+)-HCO3 (-) cotransporter NBCe1 cause proximal renal tubular acidosis (pRTA) associated with extrarenal manifestations such as ocular abnormalities and migraine. Previously, the NBCe1 cytosolic mutant S982NfsX4 was shown to have a dominant negative effect by forming hetero-oligomer complexes with wild type (WT), which might be responsible for the occurrence of glaucoma and migraine in the heterozygous family members. In this study, we investigated whether the NBCe1 L522P mutant has a similar dominant negative effect. Functional analyses in Xenopus oocytes and HEK293 cells revealed that the L522P mutant had no transport activity due to defective membrane expression. Furthermore, when coexpressed with WT, L522P significantly reduced the transport activity of WT. In HEK293 cells, the cytosolic mutant L522P reduced the membrane expression of NBCe1 by forming hetero-oligomer complexes with WT. Among the artificial Leu(522) mutants, L522I showed proper membrane expression and normal transport activity. However, the other mutants L522R, L522K, L522D, and L522E showed a predominant cytosolic retention. Moreover, L522R had a dominant negative effect, when coexpressed with WT. These results indicate that Leu(522) plays an important role in the structure and trafficking of NBCe1. They also suggest that the NBCe1 mutants retaining in cytoplasm may have the dominant negative effect in common, which may induce some clinical manifestations.

A Quartet of Leucine Residues in the Guanylate Kinase Domain of CaVβ Determines the Plasma Membrane Density of the CaV2.3 Channel

Ca(V)β subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca(V)2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca(V)2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca(V)β (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca(V)2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, β10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca(V)2.3 channels. Furthermore, the normalized protein density of Ca(V)2.3 was nearly abolished with the quadruple Ca(V)β3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca(V)β3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca(V)2.3. This interaction appears to play an essential role in conferring Ca(V)β-induced modulation of the protein density of Ca(V)α1 subunits in Ca(V)2 channels.

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.

Beta-branched residues adjacent to GG4 motifs promote the efficient association of glycophorin a transmembrane helices

Protein transmenembrane (TM) segments participating in helix-helix packing commonly contain small residue patterns (termed GG4 or "small-xxx-small" motifs) at i and i + 4 positions. Within many TM segments - such as the glycophorin A (GpA) sequence L75IxxGVxxGVxxT87- the G17y-xxx-Gly83 motif often occurs in combination with large, usually beta3-branched aliphatic residues at adjacent positions, typified here by Val30 and Val84 residues. To explore the importance of local P-branched character on GpA dimerization, we made systematic replacements to all 16 combinations of single or double Ile, Leu, and AIa residues at GpA TM Val/Val positions 80 and 84. Using the TOXCAT system to assay self-oligomerization in the Escherichia coli inner membrane--we observed that (i) combinations of Val and lie residues maintained, or improved dimerization levels; (ii) single Ala or Leu mutant combinations with Val or Ile maintained near-wild type dimerization affinities; and (iii) in the absence of beta-branching, i.e., Leu/Leu, Ala/Ala and Ala/Leu combinations, GpA dimerization was significantly diminished. An apparent capacity of lle-containing mutants to increase GpA dimerization versus WT likely arises from improved van der Waals packing (vs. Val) within the locus of helix contact, consistent with correlations we noted in lipid accessibility measurements. Examination of several synthetic peptides with sequences corresponding to selected GpA mutants (VV VI, IV II, and LL) confirmed their dimerization on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The overall results reinforce the importance of a beta-branch-containing "ridge" residue to complement a "small-xxx-small groove" in promotion of TM-TM interactions.

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the beta-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the "closed" conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity.

The L-, N-, and T-type triple calcium channel blocker benidipine acts as an antagonist of mineralocorticoid receptor, a member of nuclear receptor family

Aldosterone-induced activation of mineralocorticoid receptor, a member of the nuclear receptor family, results in increased tissue damage such as vascular inflammation and cardiac and perivascular fibrosis. Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for hypertension and angina. Benidipine exhibits pleiotropic pharmacological features such as renoprotective and cardioprotective effects through triple blockade of L-, N-, and T-type calcium channels. However, the mechanism of additional beneficial effects on end-organ damage is poorly understood. Here, we examined the effects of benidipine and other calcium channel blockers on aldosterone-induced mineralocorticoid receptor activation using luciferase reporter assay system. Benidipine showed more potent activity than efonidipine, amlodipine, or azelnidipine. Benidipine depressed the response to higher concentrations of aldosterone, whereas pretreatment of eplerenone, a steroidal mineralocorticoid receptor antagonist, did not. Binding studies using [ 3H] aldosterone indicated that benidipine and other calcium channel blockers competed for binding to mineralocorticoid receptor. Benidipine and other calcium channel blockers showed antagonistic activity on Ser810 to Leu mutant mineralocorticoid receptor, which is identified in patients with early-onset hypertension. On the other hand, eplerenone partially activated the mutant. Results of analysis using optical isomers of benidipine indicated that inhibitory effect of aldosterone-induced mineralocorticoid receptor activation was independent of its primary blockade of calcium channels. These results suggested that benidipine directly inhibits aldosterone-induced mineralocorticoid receptor activation, and the antagonistic activity might contribute to the drug's pleiotropic pharmacological features.

Structural Plasticity and Functional Implications of Internal Cavities in Distal Mutants of Type 1 Non-Symbiotic Hemoglobin AHb1 fromArabidopsis thaliana

The increasing number of nonsymbiotic plant hemoglobins discovered in genomic studies in the past decade raises intriguing questions about their physiological role. Among them, the nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana deserves particular attention, as it combines an extremely high oxygen affinity with an internal hexacoordination of the distal histidine HisE7 to the heme iron in the absence of exogenous ligands. In order to gain insight into the structure-function relationships of the protein, the ligand binding properties of mutants of two conserved residues of the distal cavity, HisE7 --> Leu and PheB10 --> Leu, were investigated by experimental and computational studies and compared to results determined for the wild type (wt) protein. The Fe(2+)-deoxy HisE7 --> Leu mutant exists, as expected, in the pentacoordinated form, while a mixture of penta- and hexacoordinated forms is found for the PheB10 --> Leu mutant, with an equilibrium shifted toward the pentacoordinated form with respect to the wt protein. Spectroscopic studies of the complexes of CO and CN(-) with AHb1 and its mutants show a subtle interplay of steric and electrostatic effects by distal residues on the ligand binding to the heme. Moreover, stopped-flow and flash photolysis experiments reveal substantial kinetic differences triggered by those mutations, which are particularly manifested in the enhanced geminate rebinding and bimolecular association rate. These findings are discussed in light of the drastic alterations found by molecular dynamics simulations in the nature and distribution of internal cavities in the protein matrix of the mutants, revealing an extremely large sensitivity of the protein structure to changes in distal HisE7 and PheB10 residues. Overall, data are consistent with the putative NO-dioxygenase activity attributed to AHb1.

Analysis of Core Packing in a Cooperatively Folded Miniature Protein: The Ultrafast Folding Villin Headpiece Helical Subdomain

The helical subdomain of the villin headpiece is the smallest naturally occurring cooperatively folded protein. Its small size, simple three-helix topology, and very rapid folding have made it an extremely popular model system for computational and theoretical studies of protein folding. The domain has a well-packed hydrophobic core comprised in part of an unusual set of three closely packed phenylalanine residues, F47, F51, and F58 (denoted using the numbering of the larger headpiece protein). Aromatic-aromatic interactions have been thought to play a critical role in specifying the subdomain fold and have been proposed to play a general role in stabilizing small proteins. The modest stability of the subdomain has hindered studies of core packing since multiple mutations can lead to constructs which fail to fold, and even single mutants can result in poorly folded variants. Using a previously characterized hyperstable mutant of the domain, generated by targeting surface residues, a complete set of single, double, and triple core Phe to Leu mutants were characterized. A highly conserved surface Trp which is part of a Trp-Pro interaction was also examined. All mutants are well-folded as judged by CD and NMR, and all exhibit sigmoidal urea and thermally induced unfolding transitions, thus proving that aromatic-aromatic, aromatic-proline, or aromatic-hydrophobic interactions are not required for specifying the subdomain fold. Double mutant cycle analysis demonstrates that F47 and F51 make the strongest pairwise interaction. Mutations which lack F58 are the most destabilized, although even the triple mutant is folded. Interestingly, mutation of the central Phe, F51, has the smallest effect on stability even though it makes contact with both F47 and F58 and appears to form the strongest pairwise interaction.

Crystal Structure of a New Cyan Fluorescent Protein and Its Hue-Shifted Variants,

Green fluorescent protein (GFP) based techniques are well established in molecular biology; however, the detailed mechanism for the fine-tuning of fluorescent colors remains unclear. Here, we report the cloning and crystal structure of a new cyan-emitting GFP-like protein, KCy. We also developed a mutant protein with a high folding efficiency (KCy-G4219: lambda(abs) = 453 nm; lambda(em) = 486 nm). X-ray diffraction analysis revealed that the KCy chromophore is formed from an internal Ser62-Tyr63-Gly64 tripeptide. The serine residue at the first position of the chromophore-forming tripeptide has a short polar chain (-OH) that forms a noncovalent interaction with the His38 imidazole at a distance of 2.96 A. Substitution of His38 in KCy-G4219 with Gln (KCy-R1) or Leu residues resulted in a slight but significant red shift of the emission peak maximum from 486 to 492 or 496 nm, respectively. The crystal structure of KCy-R1 determined at a resolution of 1.58 A showed that the noncovalent interaction between Ser62-OH and the substituted Gln38 occurred over a longer distance (3.07 A) than that observed in the wild-type KCy. Such an interaction is absent in the Leu mutant, suggesting that this interaction is one of the key factors responsible for fine-tuning the emission peak maxima, which are affected by chromophore polarization. Moreover, the structural comparison suggests that an additional water molecule buried in the space between the Ala158 residue and the chromophore phenolate is also responsible for the chromophore polarization.

Isolation and Characterization of mAMSA-hypersensitive Mutants: CYTOTOXICITY OF Top2 COVALENT COMPLEXES CONTAINING DNA SINGLE STRAND BREAKS

Topoisomerase II (Top2) is the primary target for active anti-cancer agents. We developed an efficient approach for identifying hypersensitive Top2 mutants and isolated a panel of mutants in yeast Top2 conferring hypersensitivity to the intercalator N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide (mAMSA). Some mutants conferred hypersensitivity to etoposide as well as mAMSA, whereas other mutants exhibited hypersensitivity only to mAMSA. Two mutants in Top2, changing Pro(473) to Leu and Gly(737) to Val, conferred extraordinary hypersensitivity to mAMSA and were chosen for further characterization. The mutant proteins were purified, and their biochemical activities were assessed. Both mutants encode enzymes that are hypersensitive to inhibition by mAMSA and other intercalating agents and exhibited elevated levels of mAMSA-induced Top2:DNA covalent complexes. While Gly(737) --> Val Top2p generated elevated levels of Top2-mediated double strand breaks in vitro, the Pro(473) --> Leu mutant protein showed only a modest increase in Top2-mediated double strand breaks but much higher levels of Top2-mediated single strand breaks. In addition, the Pro(473) --> Leu mutant protein also generated high levels of mAMSA-stabilized covalent complexes in the absence of ATP. We tested the role of single strand cleavage in cell killing with alleles of Top2 that could generate single strand breaks, but not double strand breaks. Expression in yeast of a Pro(473) --> Leu mutant that could only generate single strand breaks conferred hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2-targeting agents can be an important component of cell killing by Top2-targeting drugs.

Mutagenesis of the crystal contact of acidic fibroblast growth factor.

An attempt has been made to improve a crystal contact of human acidic fibroblast growth factor (haFGF; 140 amino acids) to control the crystal growth, because haFGF crystallizes only as a thin-plate form, yielding crystals suitable for X-ray but not neutron diffraction. X-ray crystal analysis of haFGF showed that the Glu81 side chain, located at a crystal contact between haFGF molecules, is in close proximity with an identical residue related by crystallographic symmetry, suggesting that charge repulsion may disrupt suitable crystal-packing interactions. To investigate whether the Glu residue affects the crystal-packing interactions, haFGF mutants in which Glu81 was replaced by Ala, Val, Leu, Ser and Thr were constructed. Although crystals of the Ala and Leu mutants were grown as a thin-plate form by the same precipitant (formate) as the wild type, crystals of the Ser and Thr mutants were grown with increased thickness, yielding a larger overall crystal volume. X-ray structural analysis of the Ser mutant determined at 1.35 A resolution revealed that the hydroxy groups of Ser are linked by hydrogen bonds mediated by the formate used as a precipitant. This approach to engineering crystal contacts may contribute to the development of large protein crystals for neutron crystallography.

Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function

Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t 1/2 of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.

Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in LivingSaccharomyces cerevisiaeCells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).

Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone

We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.

Glutamates 99 and 107 in Transmembrane Helix III of Subunit I of Cytochrome bd Are Critical for Binding of the Heme b595-d Binuclear Center and Enzyme Activity

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b558 (T187L) or b595-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b595-d center, as reported for heme b595 ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.