Effects of conformational constraint in 2- and 8-cycloleucine analogues of oxytocin and [1-penicillamine] oxytocin examined by circular dichroism and biosassay

The analogues of oxytocin and [1-penicillamine]oxytocin, containing a cycloleucine (Cle) residue in position 2 or 8, were investigated by means of circular dichroism measurements in different solvents, and the results examined in terms of their biological activities. A cycloleucine residue in position 2 substantially reduces the free conformational space of the hormone 20-membered ring moiety (including the disulfide group), and stabilizes a conformation which is close to one of the possible conformations of oxytocin and involves a -turn. In position 8, the Cle residue affects the conformation of the Tyr² side chain, apparently forcing it away from the space above the 20-membered disulfide ring. However, it does not appear that the Cle residue has any significant effect on the overall backbone conformation of the hormone. The steric effect of the penicillamine residue in position 1 on the conformation of the disulfide group and Tyr² side chain from previous investigations is further confirmed. The synthesis and biological potency of [1-penicillamine, 8-cycloleucine]oxytocin is described. This analogue exhibits a strong inhibitory effect on the uterotonic activity of oxytocinin vitro. It also inhibited the vasopressor response to vasopressin.     

Conformational Aspects of Differences in Requirements for Oxytocin and Vasopressin Receptors

Abstract

Conformational energy calculations were carried out on three non-peptide antagonists of oxytocin and vasopressin: penicilide (compound 1; selective for oxytocin receptors), 1- {1-[4-(3-acetylaminopropoxy (benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-quinoline (compound 2; selective for vasopressin V₁ receptors) and 5-dimethylamino-1-{(2-methylbenzylamino)-benzoyl}-2,3,4,5–tetrahydro-1H-benzapine (compound 3; selective for vasopressin V₂ receptors). The obtained low-energy conformations of compound 1 were compared with low-energy conformations of oxytocin (OT) and low-energy conformations of compounds 2 and 3 were compared with low-energy conformations of arginine vasopressin (AVP). It was found that the affinity of the non-peptide antagonists and their selectivity for vasopressin and oxytocin receptors is probably connected with mimicking the aromatic rings of the Tyr² and the Phe³ residues of AVP in the case of compounds 2 and 3 and with mimicking the Tyr² residue and the Ile³ or Leu⁸ residues of OT by the outer benzene ring and the isobutyl group of compound 1. Application of the results in the design of more potent non-peptide antagonists of OT and VP is also discussed.     

Theoretical conformational analysis of the opioid δ antagonist H-Tyr-Tic-Phe-OH and the μ agonist H-Tyr-D-Tic-Phe-NH2

A molecular mechanics study (grid search and energy minimization) of the highly δ receptor-selective δ opioid antagonist H-Tyr-Tic-Phe-OH (TIP; Tic: tetrahydroisoquinoline-3-car-boxylic acid) resulted in four low energy conformers with energies within 2 kcal/mol of that of the lowest energy structure. These four conformers contain trans peptide bonds only and represent compact structures showing various patterns of aromatic ring stacking. The centrally located Tic residue imposes several conformational constraints on the N-terminal dipeptide segment; however, the results of molecular dynamics simulations indicated that this tripeptide still shows some structural flexibility, particularly at the Phe³ residue. Analogous studies performed with the structurally related μ receptor-selective μ agonist H-Tyr-D -Tic-Phe-NH₂ resulted in low energy structures that were also compact but showed patterns of ring stacking different from those obtained with TIP. Superim-position of low energy conformers of TIP and H-Tyr-D -Tic-Phe-NH₂ revealed that the Phe³ residues of the L -Tic- and the D -Tic peptide were always located on opposite sides of the plane defined by the Tic residue, thus providing an explanation for the distinct activity profiles of the two compounds in structural terms. Attempts to demonstrate spatial overlap between the pharmacophoric moieties of low energy conformers of TIP and the nonpeptide δ antagonist naltrindole were made by superimposing either the Tyr¹ and Tic² aromatic rings and the N-terminal amino group or the Tyr¹ and Phe³ aromatic rings and the N-terminal amino group of the peptide with the corresponding aromatic rings and nitrogen atom in the alkaloid structure. In each case a low energy structure of TIP was found that showed good spatial overlap of all three specified pharmacophoric groups. These two conformers may represent candidate structures for the δ receptor-bound conformation of TIP. © 1994 John Wiley & Sons, Inc.     

Preferred solution and calculated conformations of dermorphin and analysis of structure–conformation–activity relationships in the series [A1an]-dermorphin

We describe the solution (¹H-nmr) and calculated conformations of the opiatelike peptide dermorphin and the analysis of structure–conformation–activity relationships in the series [Alaⁿ]-dermorphin. We used ¹H-nmr spectroscopy to study dermorphin and its analogs [Alaⁿ]-dermorphin (with n = 1, 2…7) dissolved in dimethylsufoxide. Conformational energy calculations using semiempirical partitioned energy function methods were then carried out on dermorphin and its [L -Ala²]-analog. Agreement between calculation and experiment is satisfying, both suggesting predominance of a type I β-turn around Pro⁶-Ser⁷ at the C-terminus and of an extended structure in the central sequence Phe³-Gly⁴-Tyr⁵. Detailed analysis by step-by-step substitutions with Ala indicates that intraresidue interactions dominate over medium-range interactions (between adjacent residues), although the latter may also have a noticeable influence in shaping conformations. As a general feature, the effects of substitutions on the arrangement of side chains are always larger on the succeeding residue than on the preceding residue. Almost all the variations of activity observed in the analogs can be explained from conformational changes occurring in the aromatic side chains of the biologically important Tyr¹, Phe³, and Tyr⁵ on substitutions effected on adjacent residues (fluctuations via medium-range interactions).     

Conformation-activity relationships of cyclic dermorphin analogues

A theoretical conformational analysis (molecular mechanics study) of nine cyclic tetrapeptides, structurally related to the highly mu-receptor-selective dermorphin analogue H-Tyr-D-Orn-Phe-Asp-NH2, was performed. These compounds display considerable diversity in their mu-receptor affinity and selectivity. A systematic search and subsequent energy minimization in absence of the exocyclic Tyr1 residue and Phe3 side chain revealed the constrained nature of the 11-13-membered ring structures contained in these analogues. No more than four low-energy conformers (within 2 kcal/mol of the lowest energy conformation) were found in each case. After attachment of the Tyr1 moiety and Phe3 side chain to the "bare" low-energy ring structures, a systematic search and energy minimization of these exocyclic moieties resulted in a limited number of low-energy conformational families for all compounds. Five analogues with high mu-receptor affinity--H-Tyr-D-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-Phe-D-Asp-NH2, H-Tyr-D-Asp-Phe-Orn-NH2, H-Tyr-D-Asp-Phe-A2bu-NH2 (A2 bu: alpha, gamma-diaminobutyric acid) and H-Tyr-D-Cys-Phe-Cys-NH2--all showed a tilted stacking interaction between the Tyr1 and Phe3 aromatic rings in the lowest or second lowest energy conformation found. The same kind of stacking was not possible in low-energy conformers of the four analogues with poor affinity for the mu-receptor [H-Tyr-L-Orn-Phe-Asp-NH2, H-Tyr-D-Orn-D-Phe-Asp-NH2, H-Tyr-D-Orn-Phe(NMe)-Asp-NH2 [Phe(NMe): N alpha-methylphenylalanine], and H-Tyr-D-Orn-Phg-Asp-NH2 (Phg: phenylglycine)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational analysis of cyclic angiotensin II analogues

Summary Conformationally restricted cyclic analogues of angiotensin II (ANG II), Asp¹-Arg²-Val³-Tyr⁴-Val⁵-His⁶-Pro⁷-Phe⁸, with a link between positions 3 and 5 have considerable biological activity. It is proposed that the spatial arrangement of the pharmacophore groups of Tyr⁴, His⁶ and Phe⁸ side chains and the C-terminal carboxyl group in ANG II and active analogues is similar. Conformational analysis of ANG II and two cyclic analogues c[Sar¹, Lys³,Glu⁵]ANG II and c[Sar¹,Hcy³,Mpt⁵]ANG II was performed, and a geometrical comparison of the low-energy conformations of these compounds allowed one to propose a model of receptor-bound conformation in terms of the spatial arrangement of the pharmacophore groups. This model is characterised by the close spatial location of the His⁶-Phe⁸ side chains and the Tyr⁴ C-terminal carboxyl group and is stabilised by the electrostatic interaction of Arg² and the C-terminal carboxyl group.Abbreviations ANG II angiotensin II - Hcy homocysteine - Mpt trans-4-mercaptoproline     

Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe²²⁵) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL₃) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe²²⁹ and Tyr¹⁹² residues were the main cleavage sites. Interestingly, the Phe²²⁵ residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL₃; however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL₃ at only the N-terminus, especially at Phe³³. CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe²²⁵ and Phe²²⁹ residues newly exposed by chymase, but did not cleave Tyr¹⁹². These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe²²⁹ and Phe²⁷⁹ of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe²⁷⁹. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe²⁷⁹. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe²⁷⁹, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe²²⁹.     

Benzomorphan alkaloids: Natural peptidomimetics of opioid peptide pharmacophores

Summary The synthesis of hybrid analogues of alkaloid nalindol acid and C-terminal fragments of opioid peptides aims to provide the final proof of the proposed topographical relation between opioid peptides and benzomorphan skeleton-containing alkaloids during interaction with receptors. The biological data indicate that the topographical requirements for Tyr¹ and Phe⁴ in opioid peptides are similar for interactions with both and receptor types.     

Conformational energy calculations of enzyme-substrate complexes of lysozyme. I. Energy minimization of monosaccharide and oligosaccharide inhibitors and substrates of lysozyme

Conformational energy calculations were performed on monosaccharide and oligosaccharide inhibitors and substrates of lysozyme to examine the preferred conformations of these molecules. A grid-search method was used to locate all of the low-energy conformational regions for N-acetyl-β-D -glycosamine (NAG), and energy minimization was then carried out in each of these regions. Three stable positions for the N-acetyl group have ben located, in two of which the plane of the amide unit is normal to the mean plane of the pyranosyl ring. Nine local energy minima were located for the —CH₂OH group. The positions of the two vicinal cis —OH groups are determined predominantly by interactions with either the —CH₂OH or the N-acetyl group. The most stable conformations of β-N-acetylmuramic acid (NAM) were determined from the study of the low-energy conformations of NAG. In the two stable orientations for the D -lactic acid side chain, the O—C—C′ plane (C′ being the carbon atom of the terminal carboxyl group) was found to be normal to the mean plane of the pyranosyl ring. The low-energy positions for the COOH group of NAM are determined mainly by interactions with neighboring groups. The conformational preferences of the α-anomers of NAG and NAM were also explored. The calculated conformation of the N-acetyl group for α-NAG was quite close to that determined by X-ray analysis. Two of the three lowest energy conformations of α-NAM are similar to the corresponding conformations of the β-anomer. A third low-energy structure, which has a hydrogen bond from the NH of the N-acetyl group to the C?O of the lactic acid group, corresponds very closely to the X-ray structure of this molecule. The preferred conformations of the disaccharides NAG–NAG, NAM–NAG and NAG–NAM were also investigated. Two preferred orientations of the reducing pyranosyl ring relative to the nonreducing ring were found for all of these disaccharides, both of which are close to the extended conformation. In one of these conformations, a hydrogen bond can form between the OH group attached to C₃ of the reducing sugar and the ring oxygen of the preceding residue. Each conformation can be stabilized further by a hydrogen bond between the CH₂OH (donor) of residue i + 1 and the C?O of residue i (acceptor). The interactions that determine conformations for all oligosaccharides containing both NAG and NAM are shown to be exclusively intraresidue and nearest neighbor interactions, so that it is possible to predict all stable conformations of oligosaccharides containing NAG and NAM in any sequence.     

Four-component pharmacophore model for endomorphins toward μ opioid receptor subtypes

In the present work, a series of simulation tools were used to determine structure-activity relationships for the endomorphins (EMs) and derive μ-pharmacophore models for these peptides. Potential lowest energy conformations were determined in vacuo by systematically varying the torsional angles of the Tyr¹-Pro² (ω₁) and Pro²-Trp³/Phe³ (ω₂) as tuning parameters in AM1 calculations. These initial models were then exposed to aqueous conditions via molecular dynamics simulations. In aqueous solution, the simulations suggest that endomorphin conformers strongly favor the trans/trans pair of the ω₁/ω₂ amide bonds. From two-dimensional probability distributions of the ring-to-ring distances with respect to the pharmacophoric angles for EMs, a selectivity range of μ₁ is ca. 8.3 ~ 10.5 Å for endomorphin-2 and selectivity range of μ₂ is ca. 10.5 ~ 13.0 Å for endomorphin-1 were determined. Four-component μ-pharmacophore models are proposed for EMs and are compared to the previously published δ- and κ-pharmacophore models.

Angle NAB/C vs distance

     

Conformational characteristics of polyglutamic acid esters: Persistence of orientational correlation along the side chain flanking the α-helical backbone

The side chain conformations of α-helical poly(L -glutamic acid) esters $ \rlap{--}[NHCH(CH_2 CH_2 COOR)CO\rlap{--}]_x $, carrying a homologous series of ester residues such as R = ? (CH₂)_n? with n = 1–3, have been studied in the lyotropic liquid crystalline state (chloroform 20 v/v%) by the deuterium nmr method. In order to study the surface chirality of the molecule, the phenyl groups situated at the terminal of the side chain have been deuterated. From the observed deuterium quadrupolar splittings, the average inclination θ_p of the para-axis of the phenyl group with respect to the α-helical backbone was elucidated. A distinct odd–even oscillation in the quantity such as 〈 cos² θ_p〉 was observed with the number of methylene units n. A rotational isomeric state analysis has indicated that the observed orientational correlation arises from the interdependence of the neighboring bond rotation along the side chain. Preference of the “extended” conformations is also enhanced by the mutual conformational exclusion of neighboring side chains.     

Aromatic Anchor at an Invariant Hormone-Receptor Interface: FUNCTION OF INSULIN RESIDUE B24 WITH APPLICATION TO PROTEIN DESIGN*

Crystallographic studies of insulin bound to fragments of the insulin receptor have recently defined the topography of the primary hormone-receptor interface. Here, we have investigated the role of Phe^B24, an invariant aromatic anchor at this interface and site of a human mutation causing diabetes mellitus. An extensive set of B24 substitutions has been constructed and tested for effects on receptor binding. Although aromaticity has long been considered a key requirement at this position, Met^B24 was found to confer essentially native affinity and bioactivity. Molecular modeling suggests that this linear side chain can serve as an alternative hydrophobic anchor at the hormone-receptor interface. These findings motivated further substitution of Phe^B24 by cyclohexanylalanine (Cha), which contains a nonplanar aliphatic ring. Contrary to expectations, [Cha^B24]insulin likewise exhibited high activity. Furthermore, its resistance to fibrillation and the rapid rate of hexamer disassembly, properties of potential therapeutic advantage, were enhanced. The crystal structure of the Cha^B24 analog, determined as an R₆ zinc-stabilized hexamer at a resolution of 1.5 Å, closely resembles that of wild-type insulin. The nonplanar aliphatic ring exhibits two chair conformations with partial occupancies, each recapitulating the role of Phe^B24 at the dimer interface. Together, these studies have defined structural requirements of an anchor residue within the B24-binding pocket of the insulin receptor; similar molecular principles are likely to pertain to insulin-related growth factors. Our results highlight in particular the utility of nonaromatic side chains as probes of the B24 pocket and suggest that the nonstandard Cha side chain may have therapeutic utility.     

Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Abstract

Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R₁ and MCH‐R₂, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R₁ was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R₁ and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R₁ (SLC‐1), the radioligand was [¹²⁵I]–[Tyr¹³]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [¹²⁵I]–[D‐Phe¹³, Tyr¹⁹]‐MCH served as radioligand. The bioassay used for MCH‐R₁ was intracellular Ca²⁺ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R₁ and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe¹³ in [Phe¹³, Tyr¹⁹]‐MCH was well tolerated by both MCH‐R₁ and B16 MCH‐R, change of configuration to D‐Phe¹³ in [D‐Phe¹³, Tyr¹⁹]‐MCH or [D‐Phe¹³]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R₁. By contrast, the D‐Phe¹³ residue increased the affinity of [D‐Phe¹³, Tyr¹⁹]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe¹³, Tyr¹⁹]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R₁ in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R₁.     

Nociceptin and its natural and specifically‐modified fragments: Structural studies

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically‐modified [Tyr¹]FQ (1‐6), [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel β‐sheet conformation. FQ (1‐11), FQ (7‐17) [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) have an α‐helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ～8.3) was investigated by means of surface‐enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7‐17), chemisorbed on the colloidal silver surfaces through a Phe⁴ residue, which for FQ, FQ (1‐11), FQ (1‐6), [Tyr¹]FQ (1‐6), and [His¹]FQ (1‐6) lies almost flat on this surface, while for FQ (1‐13) and FQ (1‐13)NH₂ adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1‐6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe⁴ side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe⁴ in [His¹]FQ (1‐6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1‐13) and FQ (7‐17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the ? NH₂ group. We also showed that upon adsorption, the secondary structure of these peptides is altered. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1039–1054, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Solution structures of staphylococcal nuclease from multidimensional, multinuclear NMR: Nuclease-H124L and its ternary complex with Ca2+ and thymidine-3′,5′-bisphosphate

The solution structures of staphylococcal nuclease (nuclease) H124L and itsternary complex, (nuclease-H124L)pdTpCa²⁺, were determinedby ab initio dynamic simulated annealing using 1925 NOE, 119 , 20¹ and 112 hydrogen bond constraints for the free protein,and 2003 NOE, 118 , 20 ¹ and 114 hydrogen bondconstraints for the ternary complex. In both cases, the final structuresdisplay only small deviations from idealized covalent geometry. In structuredregions, the overall root-mean-square deviations from mean atomic coordinatesare 0.46 (±0.05) Å and 0.41 (±0.05) Å for thebackbone heavy atoms of nuclease and its ternary complex, respectively. Thebackbone conformations of residues in the loop formed byArg⁸¹–Gly⁸⁶, which is adjacent to the activesite, are more precisely defined in the ternary complex than in unligatednuclease. Also, the protein side chains that show NOEs and evidence forhydrogen bonds to pdTp (Arg³⁵, Lys⁸⁴,Tyr⁸⁵, Arg⁸⁷, Tyr¹¹³, andTyr¹¹⁵) are better defined in the ternary complex. As has beenobserved previously in the X-ray structures of nuclease-WT, the binding ofpdTp causes the backbone of Tyr¹¹³ to change from an extendedto a left-handed -helical conformation. The NMR structures reportedhere were compared with available X-ray structures: nuclease-H124L [Truckseset al. (1996) Protein Sci., 5, 1907–1916] and the ternary complex ofwild-type staphylococcal nuclease [Loll and Lattman (1989) Proteins Struct.Funct. Genet., 5, 183–201]. Overall, the solution structures ofnuclease-H124L are consistent with these crystal structures, but smalldifferences were observed between the structures in the solution and crystalenvironments. These included differences in the conformations of certain sidechains, a reduction in the extent of helix 1 in solution, and many fewerhydrogen bonds involving side chains in solution.     

Energetics and proton release in photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA2 or psbA3 gene

In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for the Photosystem II (PSII) D1 subunit that interacts with most of the main cofactors involved in the electron transfers. Recently, the 3D crystal structures of both PsbA2-PSII and PsbA3-PSII have been solved [Nakajima et al., J. Biol. Chem. 298 (2022) 102668.]. It was proposed that the loss of one hydrogen bond of Phe_D1 due to the D1-Y147F exchange in PsbA2-PSII resulted in a more negative E_m of Phe_D1 in PsbA2-PSII when compared to PsbA3-PSII. In addition, the loss of two water molecules in the Cl-1 channel was attributed to the D1-P173M substitution in PsbA2-PSII. This exchange, by narrowing the Cl-1 proton channel, could be at the origin of a slowing down of the proton release. Here, we have continued the characterization of PsbA2-PSII by measuring the thermoluminescence from the S₂Q_A⁻/DCMU charge recombination and by measuring proton release kinetics using time-resolved absorption changes of the dye bromocresol purple. It was found that i) the E_m of Phe_D1^–/Phe_D1 was decreased by ∼30 mV in PsbA2-PSII when compared to PsbA3-PSII and ii) the kinetics of the proton release into the bulk was significantly slowed down in PsbA2-PSII in the S₂Tyr_Z^• to S₃Tyr_Z and S₃Tyr_Z^• → (S₃Tyr_Z^•)’ transitions. This slowing down was partially reversed by the PsbA2/M173P mutation and induced by the PsbA3/P173M mutation thus confirming a role of the D1-173 residue in the egress of protons trough the Cl-1 channel.