Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.     

Conformational stability of antamanide and analogs. Crystal structure of perhydrosymmetric antamanide, cyclic (-Val-Pro-Pro-Cha-Cha-Val-Pro-Pro-Cha-Cha-)

The synthetic perhydrogenated symmetric analog of the cyclic decapeptide antamanide is biologically inactive, although the conformation of the molecule and the crystal packing are very similar to that of the active symmetric analog of antamanide. In fact, the same conformation for the molecule has now been found in six polymorphs of uncomplexed antamanide and its analogs. The differences between the active and inactive antamanide analogs are displayed dramatically in the conformations of their metal ion (Na+ or Li+) complexes, thus suggesting strongly that for physiological activity antamanide is not in the conformation assumed by the uncomplexed molecule, but rather in the conformation assumed by the complexed state of natural antamanide. The present structure crystallizes in space group P2(1)2(1)2(1) with a = 20.515(14) A, b = 21.316(16) A, c = 17.128(16) A and four peptide molecules in the unit cell. There are three cocrystallized water molecules at full occupancy intrinsic to the peptide, and several more water molecules or other solvent molecules at partial occupancy. The formula of the peptide is C66H106N10O10.4-H2O.2X.     

Mutation of Tyr138 disrupts the structural coupling between the opposing domains in vertebrate calmodulin

We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.     

Conformation of acetylcholine receptor in the presence of agonists and antagonists

The conformations of acetylcholine receptor fromTorpedo californica in the absence and presence of agonists, antagonists, and local anesthetics were studied by circular dichroism (CD). Without ligands, the receptor had about 40% helix, 20% -sheets, and 10% -turns as analyzed from its far-UV CD spectrum. Its near-UV CD spectrum resembled that of acetylcholinesterase from the same source. None of the ligands studied altered the far-UV spectrum of the receptor. However, in the near-UV region, carbamylcholine and acetylcholine shifted the Phe and Tyr bands of AChR to less negative, whereas hexamethonium changed the Tyr bands to more negative, indicating that the site of binding of agonists and antagonists and their effect on the conformation of the receptor may be different. Decamethonium, procaine, and lidocaine had no effect on both the far- and near-UV CD spectra of acetylcholine receptor.     

Luminescence study of G-quadruplex formation in the presence of Tb3+ ion

The interactions of Tb3+ with the quadruplex-forming oligonucleotide bearing human telomeric repeat sequence d(G(3)T(2)AG(3)T(2)AG(3)T(2)AG(3)), (htel21), have been studied using luminescence spectroscopy and circular dichroism (CD). Enhanced luminescence of Tb3+, resulting from energy transfer from guanines, indicated encapsulation of Tb3+ ion in the central cavity of quadruplex core. The ability of lanthanide ions (Eu3+ and Tb3+) to mediate formation of quadruplex structure has been further evidenced by the fluorescence energy transfer measurements with the use of oligonucleotide probe labeled with fluorescein and rhodamine FRET partners, FAM-htel21-TAMRA. The CD spectra revealed that Tb3+/htel21 quadruplex possesses antiparallel strand orientation, similarly as sodium quadruplex. Tb3+ binding equilibria have been investigated in the absence and the presence of competing metal cations. At low Tb3+ concentration (8 microM) Tb3+/htel21 quadruplex stability is very high (5 x 10(6) M(-1)) and stoichiometry of 5-7 Tb3+ ions per one quadruplex molecule is observed. Luminescence and CD titration experiments suggested that the cavity of quadruplex accommodates two Tb3+ ions and the remaining Tb3+ ions bind probably to TTA loops of quadruplex. Higher concentration of Tb3+ (above 10 microM) results in the excessive binding of Tb3+ ions that finally destabilizes quadruplex, which undergoes transformation into differently organized assemblies. Such assemblies (probably possessing multiple positive charge) exhibit kinetic stability, which is manifested by a very slow kinetics of displacement of Tb3+ ion by competing cations (Li+, Na+, K+).     

Cyclic enkephalin analogs containing various para-substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists.

The cyclic enkephalin analog H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) is a highly potent opioid agonist with IC(50)s of 35 pm and 19 pm in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe(1)-analog of this peptide showed 370-fold and 6790-fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr(1) hydroxyl-group in the interaction with mu and delta opioid receptors. In the present study, the effect of various substituents (-NH(2), -NO(2), -CN, -CH(3), -COOH, -COCH(3), -CONH(2)) introduced in the para-position of the Phe(1)-residue of H-Phe-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) on the in vitro opioid activity profile was examined. Most analogs showed enhanced mu and delta agonist potencies in the two bioassays, except for the Phe(pCOOH)(1)-analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH(3))(1)- and the Phe(pCONH(2))(1)-analogs. The latter compound showed subnanomolar mu and delta agonist potencies and represents the most potent enkephalin analog lacking the Tyr(1) hydroxyl-group reported to date. Taken together, these results indicate that various substituents introduced in the para-position of Phe(1) enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure-activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.     

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides,cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Complex formation with alkali and alkaline earth metal ions of cyclic octapeptides, cyclo(Phe-Pro)₄, cyclo(Leu-Pro)₄, and cyclo[Lys(Z)-Pro]₄ was investigated in relation to conformation. In an alcohol solution, cyclo(Phe-Pro)₄ did not form complexes. However, cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ formed complexes selectively with Ba²⁺ and Ca²⁺ ions. Changing the solvent from alcohol to acetonitrile, the complexation behavior was very different. In acetonitrile, cyclo(Phe-Pro)₄ was found to form a complex with Ba²⁺, and CD spectra of cyclo(Leu-Pro)₄ and cyclo[Lys(Z)-Pro]₄ changed sharply on complexation with K⁺. Rate constants of the complex formation between the cyclic octapeptides and metal salts were in the range of 0.7–12 L mol^?1 min^?1 in an alcohol solution. One of the two types of complex formation in acetonitrile was much faster than that in an alcohol solution.     

Conformational and ion-binding properties of cyclolinopeptide A isolated from linseed

The conformation of the cyclic nonaPePtide from linseed, cyclolinoPePtide A in methanol and in acetonitrile has been elucidated by one-and two-dimensional nuclear magnetic resonance. The molecule is folded in a β-turn conformation. CyclolinoPePtide A interacts and weakly comPlexes with Tb³⁺ (a Ca²⁺ mimic ion) with the metal ion Positioned Proximally to the Phe residue, but with no substantial structural alteration uPon metal binding. CyclolinoPePtide A is also seen to aid the translocation of Pr³⁺ (another Ca²⁺ mimic) across unilamellar liPosomes. However, cyclolinoPePtide A does not Phase transfer or act as an ionoPhore of calcium ion myself. ExPeriments using lanthanide ions thus do not necessarily indicate any ionoPhoretic ability of the comPlexone towards calcium ions.     

Design,synthesis, and opioid activity of arodyn analogs cyclized by ring-closing metathesis involving Tyr(allyl)

Kappa (κ) opioid receptor selective antagonists are useful pharmacological tools in studying κ opioid receptors and have potential to be used as therapeutic agents for the treatment of a variety of diseases including mood disorders and drug addiction. Arodyn (Ac[Phe^1–3,Arg⁴,d-Ala⁸]Dyn A-(1–11)NH₂) is a linear acetylated dynorphin A (Dyn A) analog that is a potent and selective κ opioid receptor antagonist (Bennett et al. J Med Chem 2002;45:5617–5619) and prevents stress-induced reinstatement of cocaine-seeking behavior following central administration (Carey et al. Eur J Pharmacol 2007;569:84–89). To restrict its conformational mobility, explore possible bioactive conformations and potentially increase its metabolic stability we synthesized cyclic arodyn analogs on solid phase utilizing a novel ring-closing metathesis (RCM) reaction involving allyl-protected Tyr (Tyr(All)) residues. This approach preserves the aromatic functionality and directly constrains the side chains of one or more of the Phe residues. The novel cyclic arodyn analog 4 cyclized between Tyr(All) residues incorporated in positions 2 and 3 exhibited potent κ opioid receptor antagonism in the [³⁵S]GTPγS assay (K_B?=?3.2?nM) similar to arodyn. Analog 3 cyclized between Tyr(All) residues in positions 1 and 2 also exhibited nanomolar κ opioid receptor antagonist potency (K_B?=?27.5?nM) in this assay. These are the first opioid peptides cyclized via RCM involving aromatic residues, and given their promising pharmacological activity represent novel lead peptides for further exploration.     

Conformational stability of CopC and roles of residues Tyr79 and Trp83

CopC is a periplasmic copper Chaperone protein that has a β‐barrel fold and two metal‐binding sites distinct for Cu(II) and Cu(I). In the article, four mutants (Y79F, Y79W, Y79WW83L, Y79WW83F) were obtained by site‐directed mutagenesis. The far‐UV CD spectra of the proteins were similar, suggesting that mutations did not bring any significant changes in secondary structures. Meanwhile the effects of mutations on the protein's function were manifested by Cu(II) binding. Fluorescence lifetime measurement and quenching of tryptophan fluorescence by acrylamide and KI showed that the microenvironment around Trp83 was more hydrophobic than that around Tyr79 in apoCopC. Unfolding experiments induced by guanidinium chloride (GdnHCl), urea provided the conformational stability of each protein. The Δ<ΔG⁰_element> obtained using the model of structural elements was used to show the role of Tyr79 and Trp83. On the one hand, the <ΔG⁰_element> induced by urea for Y79F, Y79W have a loss of 6.51, 2.03 kJ/mol, respectively, compared with apoCopC, proving that replacement of Tyr79 by Phe or Trp all decreased the protein stability, meaning that the hydrogen bonds interactions between Tyr79 and Thr75 played an important role in stabilizing apoCopC. On the other hand, the <ΔG⁰_element> induced by urea for Y79WW83L have a loss of 11.44 kJ/mol, but for Y79WW83F did a raise of 1.82 kJ/mol compared with Y79W. The replacement of Trp83 by Phe and Leu yields opposite effects on protein stability, which suggested that the aromatic ring of Trp83 was important in maintaining the hydrophobic core of apoCopC.     

Conformational constraints in angiotensin IV to probe the role of Tyr2, Pro5 and Phe6

The aromatic amino acids Tyr and Phe in angiotensin IV (Ang IV) were conformationally constrained by the use of β‐Me substituted analogs, or cyclic constrained analogs. None of these modifications was allowed for Tyr¹, while only e‐β‐MePhe⁶ substitution resulted in an AngIV analog with high IRAP potency and selectivity versus AP‐N or the AT₁ receptor. This indicates an important role of the orientation of the Phe⁶ for inducing selectivity. Pro⁵ replacement with 2‐aminocyclopentanecarboxylic acid maintained IRAP potency and abolished AT₁ affinity. These results confirm the importance of conformational constrained amino acids to generate selectivity in bioactive peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Antamanide, a derivative of Amanita phalloides, is a novel inhibitor of the mitochondrial permeability transition pore

Antamanide is a cyclic decapeptide derived from the fungus Amanita phalloides. Here we show that antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by antamanide is not additive with the cyclophilin D-binding drug cyclosporin A, (ii) the inhibitory action of antamanide on the pore requires phosphate, as previously shown for cyclosporin A; (iii) antamanide is ineffective in mitochondria or cells derived from cyclophilin D null animals, and (iv) abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability transition pore inhibition by antamanide needs two critical residues in the peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on the pore in a cyclophilin D-independent fashion. Antamanide also abrogates mitochondrial depolarization and the ensuing cell death caused by two well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal peptide. Our findings have implications for the comprehension of cyclophilin D activity on the permeability transition pore and for the development of novel pore-targeting drugs exploitable as cell death inhibitors.     

The binding of terbium ions to gelsolin reveals two classes of metal ion binding sites.

Spectroscopically active terbium ions have been used to probe the Ca2+ ion-binding sites on human plasma gelsolin. The luminescence of Tb3+ ions bound to gelsolin is markedly enhanced when excited indirectly at 295 nm due to F?rster type dipole-dipole energy transfer from neighboring tryptophan residues. Titration of this luminescence with increasing concentrations of Tb3+ ions was saturable although the shape of this titration curve was complex indicating the involvement of multiple classes of sites. Luminescence lifetime measurements (obtained by indirect excitation at 295 nm) demonstrate the presence of two classes of sites characterized by a major lifetime of 1.0-1.1 ms and a minor lifetime of 0.7-0.8 ms. However, while the amplitude of the minor lifetime showed a hyperbolic dependence on the Tb3+ ion concentration, the amplitude of the major lifetime showed a strongly sigmoidal dependence. Different classes of Tb3+ ion binding sites can also be distinguished by the different Ca2+ ion concentrations needed to displace Tb3+ ions from these sites on gelsolin. It is proposed that the occupancy of one class of Tb3+ ion binding sites on gelsolin causes a conformational change in gelsolin which then allows a second class of cryptic Tb3+ ion binding sites to be expressed. The implications of these results in terms of the binding of Ca2+ ions to gelsolin and the regulation of the activities of gelsolin by calcium are discussed.     

Structural and biochemical characterization of a new Mg(2+) binding site near Tyr94 in the restriction endonuclease PvuII

We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.     

Conformational and ion binding properties of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2.

The conformation and ion-binding characteristics of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2, in a liphophilic solvent, acetonitrile, have been studied using CD and nmr spectroscopy. The peptide binds preferentially to divalent cations such as calcium, magnesium, and barium. The conformations of the free cyclic peptide and its calcium complex are very similar with well-defined beta- and gamma-turns. The cyclic peptide readily forms equimolar and possibly 2:1 (peptide:cation) complexes with divalent cations.     

Spectral contribution of the individual tryptophan of alphaB-crystallin: a study by site-directed mutagenesis

There are two tryptophan residues in the lens alphaB-crystallin, Trp9 and Trp60. We prepared two Trp --> Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near-ultraviolet circular dichroism (UV CD) differently. The major difference in the near-UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near-UV CD shows an increased intensity in the region of 270-280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a beta-strand.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of active‐site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C‐S lyase

In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C‐S lyase from Corynebacterium diphtheriae is a pyridoxal 5′‐phosphate‐dependent enzyme in the transsulfuration pathway that catalyzes the α,β‐elimination of sulfur‐containing amino acids, such as l ‐cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L ‐methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active‐site residues, Y55F and Y114F, were characterized by UV‐visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady‐state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130‐fold decrease in at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP‐binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of l ‐cystathionine, and causes a change in substrate specificity towards l ‐serine and O‐acetyl‐l ‐serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C‐S lyase, which seems to be regulated by active‐site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors. Proteins 2015; 83:78–90. © 2014 Wiley Periodicals, Inc.     

Conformational characteristics of peptides and unanticipated results from crystal structure analyses

Preferred conformation and types of molecular folding are some of the topics that can be addressed by structure analysis using x-ray diffraction of single crystals. The conformations of small linear peptide molecules with 2-6 residues are affected by polarity of solvent, presence of water molecules, hydrogen bonding with neighboring molecules, and other packing forces. Larger peptides, both cyclic and linear, have many intramolecular hydrogen bonds, the effect of which outweighs any intermolecular attractions. Numerous polymorphs of decapeptides grown from a variety of solvents, with different cocrystallized solvents, show a constant conformation for each peptide. Large conformational changes occur, however, upon complexation with metal ions. A new form of free valinomycin grown from DMSO exhibits near three-fold symmetry with only three intramolecular hydrogen bonds. The peptide is in the form of a shallow bowl with a hydrophobic exterior. Near the bottom of the interior of the bowl are three carbonyl oxygens, spaced and directed so that they are in position to form three ligands to a K+, e.g., complexation can be completed by the three lobes containing the beta-bends closing over and encapsulating the K+ ion. In another example, free antamanide and the biologically inactive perhydro analogue, in which four phenyl groups become cyclic hexyl groups, have essentially the same folding of backbone and side chains. The conformation changes drastically upon complexation with Li+ or Na+. However, the metal ion complex of natural antamanide has a hydrophobic globlar form whereas the metal ion complex of the inactive perhydro analogue has a polar band around the middle. The structure results indicate that the antamanide molecule is in a complexed form during its biological activity. Single crystal x-ray diffraction structure analyses have identified the manner in which water molecules are essential to creating minipolar areas on apolar helices. Completely apolar peptides, such as membrane-active peptides, can acquire amphiphilic character by insertion of a water molecule into the helical backbone of Boc-Aib-Ala-Leu-Aib-Ala-Leu-Aib-Ala-Leu-Aib-OMe, for example. The C-terminal half assumes an alpha-helix conformation, whereas the N-terminal half is distorted by an insertion of a water molecule W(1) between N(Ala5) and O(Ala2), forming hydrogen bonds N(5)H...W(1) and W(1)...O(2). The distortion of the helix exposes C = O(Aib1) and C = O(Aib4) to the outside environment with the consequence of attracting additional water molecules. The leucyl side chains are on the other side of the molecule. Thus a helix with an apolar sequence can mimic an amphiphilic helix.