Impacts of terminal (4R)-fluoroproline and (4S)-fluoroproline residues on polyproline conformation

Many interests have been focused on prolyl cis–trans isomerization which is related to protein folding and isomer-specific biochemical recognition. Since polyproline can adopt either type I (PPI) helices with all cis amide bonds or type II (PPII) helices with all trans amide bonds, it has been a valuable model to study the prolyl isomerization. Recent studies have shown that stereoelectronic effects govern the stability of PPII structure and the rate of PPII → PPI conversion. To further explore the terminal stereoelectronic effects on polyproline conformation, herein we synthesized a series of host–guest peptides in which (2S,4S)-4-fluoroproline (flp) or (2S,4R)-4-fluoroproline (Flp) residues are incorporated into the C- or N-terminal end of a peptide and studied the thermodynamic and kinetic consequences on polyproline conformation. Circular dichroism measurements revealed that inserting 4-fluoroproline residues into the C terminus of a polyproline peptide induces a great stereoelectronic effect on PPII stability and PPII → PPI conversion rates. From the C terminus, a (Flp)₃ triplet stabilizes PPII structure and increases the transition barrier of PPII → PPI conversion by 1.53 kJ mol^?1 while a (flp)₃ triplet destabilizes PPII conformation and reduce the PPII → PPI transition barrier by 4.61 kJ mol^?1. In contrast, the 4-fluoroproline substitutions at the N terminus do not exhibit distinct stereoelectronic effects on PPII stability and PPII → PPI conversion rates. Our data demonstrate that the C-terminal stereoelectronic effects have a more dramatic impact on PPII stability and PPII → PPI conversion kinetics.     

Modulating the folding stability and ligand binding affinity of Pin1 WW domain by proline ring puckering

The pyrrolidine side chain makes proline play a unique role in protein structure and function. The C^γ ring pucker preference and the cis– trans peptidyl bond ratio can be mediated via stereoelectronic effects. Here we used a compact triple‐stranded antiparallel β‐sheet protein, the human Pin1 WW domain, to study the consequences of implanting a preorganized C^γ ring pucker on protein structure and function. The conserved Pro37 is a key residue involved in one hydrophobic core, plays an important role in the WW domain, and adopts a C^γ‐endo ring pucker in the native structure. Pro37 was replaced with C^γ‐exo biased pucker derivatives: (2S,4R)‐4‐hydroxyproline (4R‐Hyp), (2S,4R)‐4‐fluoroproline (4R‐Flp), (2S,4R)‐4‐methoxyproline (4R‐Mop), and C^γ‐endo biased pucker derivatives: (2S,4S)‐4‐hydroxyproline (4S‐hyp), (2S,4S)‐4‐fluoroproline (4S‐flp), (2S,4S)‐4‐methoxyproline (4S‐mop) to examine how a preorganized pucker affects the folding stability and ligand‐binding affinity. Circular dichroism measurements indicate that among the variants, only the one with 4S‐flp substitution (P37flp) is more stable than the wild type, suggesting that the stabilization effects originated from preorganization of the backbone conformation and the hydrophobicity of C? F group. Analysis of ligand‐binding affinity using isothermal titration calorimetry revealed that only P37flp has a stronger ligand affinity than the wild type, showing that 4S‐flp can stabilize the WW domain and increase its ligand affinity. Together we have used 4‐substituted proline derivatives and the WW domain to demonstrate that proline ring puckering can be a key factor in determining the folding stability of a protein but the choice of the derivative groups is also critical. Proteins 2014; 82:67–76. © 2013 Wiley Periodicals, Inc.     

Stereoelectronic effects on polyproline conformation

The polyproline type II (PPII) helix is a prevalent conformation in both folded and unfolded proteins, and is known to play important roles in a wide variety of biological processes. Polyproline itself can also form a type I (PPI) helix, which has a disparate conformation. Here, we use derivatives of polyproline, (Pro)10, (Hyp)10, (Flp)10, and (flp)10, where Hyp is (2S,4R)-4-hydroxyproline, Flp is (2S,4R)-4-fluoroproline, and flp is (2S,4S)-4-fluoroproline, to probe for a stereoelectronic effect on the conformation of polyproline. Circular dichroism spectral analyses show that 4R electron-with-drawing substituents stabilize a PPII helix relative to a PPI helix, even in a solvent that favors the PPI conformation, such as n-propanol. The stereochemistry at C4 ordains the relative stability of PPI and PPII helices, as (flp)10 forms a mixture of PPI and PPII helices in water and a PPI helix in n-propanol. The conformational preferences of (Pro)10 are intermediate between those of (Hyp)10/(Flp)10 and (flp)10. Interestingly, PPI helices of (flp)10 exhibit cold denaturation in n-propanol with a value of T(s) near 70 degrees C. Together, these data show that stereoelectronic effects can have a substantial impact on polyproline conformation and provide a rational means to stabilize a PPI or PPII helix.     

Stereoelectronic effects on the transition barrier of polyproline conformational interconversion

There has been growing interest in polyproline type II (PPII) helices since PPII helices have been found in folded and unfolded proteins and involved in a variety of biological activities. Polyproline can also form type I helices (PPI) which are very different from PPII conformation and only exist in certain organic solvents. Recent studies have shown that stereoelectronic effects play a critical role in stabilizing a PPI or PPII helix. Here, we have synthesized a series of host–guest peptides with an electron‐withdrawing substituent at the 4R or 4S position of proline and used a kinetic approach to further explore stereoelectronic effects on the transition barrier of the interconversion between PPI and PPII conformations. Time‐dependent circular dichroism measurements revealed that the rates of PPII → PPI conversion were reduced upon incorporating the hydroxyl‐, fluoro‐, and methoxy‐groups at the 4R position while the rates would be increased if these substituents were at the 4S position. We quantified the changes in transition free energy by comparing their rate constants. (4R,2S)‐4‐Fluoroproline and (4S,2S)‐4‐fluoroproline have the largest effect on the transition energy barrier for PPII → PPI conversion. Our results provide important insights into the role of stereoelectronic effects on the PPII → PPI transition state barrier, which has not been reported in past thermodynamic studies.     

Stabilization mechanism of triple helical structure of collagen molecules

Summary The role of 4-hydroxyproline (Hyp) in stabilizing collagen triple helical structure has been investigated comprehensively. Recently it was emphasized that the preferential pyrrolidine ring pucker influenced by the stereoelectronic effects of substituted groups mainly affects the thermal stability of the triple helix. To examine this explanation, we synthesized and characterized (fPro ^R -Pro-Gly)₁₀ and (fPro ^S -Pro-Gly)₁₀. According to the results of CD and analytical ultracentrifugation, (fPro ^S -Pro-Gly)₁₀ takes a triple helical structure and (fPro ^R -Pro-Gly)₁₀ exists in a single chain structure, the trend of which is not consistent with the relationship between (Hyp ^S -Pro-Gly)₁₀ and (Hyp ^R -Pro-Gly)₁₀. In order to rationalize experimental results as a whole, we carried out DSC analyses and determined the thermodynamic parameters associated with the structural transition of these collagen model peptides. In this paper, we reported the DSC results for (Pro-Pro-Gly)₁₀, (Pro-Hyp ^R -Gly)₁₀ and (Pro-fPro ^R -Gly)₁₀ as a part of this study. Based on those parameters, we concluded that Hyp and fPro stabilize the triple helix in different stabilizing mechanisms; the increased stability of (Pro-Hyp ^R -Gly)₁₀ is ascribed primarily to the enthalpic effects while that of (Pro-fPro ^R -Gly)₁₀ is achieved through the entropic ones.     

Interstrand dipole-dipole interactions can stabilize the collagen triple helix

The amino acid sequence of collagen is composed of GlyXaaYaa repeats. A prevailing paradigm maintains that stable collagen triple helices form when (2S)-proline (Pro) or Pro derivatives that prefer the C(γ)-endo ring pucker are in the Xaa position and Pro derivatives that prefer the C(γ)-exo ring pucker are in the Yaa position. Anomalously, an amino acid sequence in an invertebrate collagen has (2S,4R)-4-hydroxyproline (Hyp), a C(γ)-exo-puckered Pro derivative, in the Xaa position. In certain contexts, triple helices with Hyp in the Xaa position are now known to be hyperstable. Most intriguingly, the sequence (GlyHypHyp)(n) forms a more stable triple helix than does the sequence (GlyProHyp)(n). Competing theories exist for the physicochemical basis of the hyperstability of (GlyHypHyp)(n) triple helices. By synthesizing and analyzing triple helices with different C(γ)-exo-puckered proline derivatives in the Xaa and Yaa positions, we conclude that interstrand dipole-dipole interactions are the primary determinant of their additional stability. These findings provide a new framework for understanding collagen stability.     

Direct and continuous assay for prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) is a nonheme iron dioxygenase that catalyzes the posttranslational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O₂ as cosubstrates, and forms succinate and CO₂ as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.     

Stabilization of collagen fibrils by hydroxyproline

The substitution of hydroxyproline for proline in position Y of the repeating Gly-X-Y tripeptide sequence of collagen-like poly(tripeptide)s (i.e., in the position in which Hyp occurs naturally) is predicted to enhance the stability of aggregates of triple helices, while the substitution of Hyp in position X (where no Hyp occurs naturally) is predicted to decrease the stability of aggregates. Earlier conformational energy computations have indicated that two triple helices composed of poly(Gly-Pro-Pro) polypeptide chains pack preferentially with a nearly parallel orientation of the helix axes [Nemethy, G., & Scheraga, H.A. (1984) Biopolymers 23, 2781-2799]. Conformational energy computations reported here indicate that the same packing arrangement is preferred for the packing of two poly(Gly-Pro-Hyp) triple helices. The OH groups of the Hyp residues can be accommodated in the space between the two packed triple helices without any steric hindrance. They actually contribute about 1.9 kcal/mol per Gly-Pro-Hyp tripeptide to the packing energy, as a result of the formation of weak hydrogen bonds and other favorable noncovalent interatomic interactions. On the other hand, the substitution of Hyp in position X weakens the packing by about 1.7 kcal/mol per Gly-Hyp-Pro tripeptide. Numerous published experimental studies have established that Hyp in position Y stabilizes an isolated triple helix relative to dissociated random coils, while Hyp in position X has the opposite effect. We propose that Hyp in position Y also enhances the stability of the assembly of collagen into microfibrils while, in position X, it decreases this stability.     

Contribution of tertiary amides to the conformational stability of collagen triple helices

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides.     

4-chloroprolines: synthesis, conformational analysis, and effect on the collagen triple helix

Collagen is an abundant, triple-helical protein comprising three strands of the repeating sequence: Xaa-Yaa-Gly. (2S)-Proline and (2S,4R)-4-hydroxyproline (Hyp) are common in the primary structure of collagen. Here, we use nonnatural proline derivatives to reveal determinants of collagen stability. Specifically, we report high-yielding syntheses of (2S,4S)-4-chloroproline (clp) and (2S,4R)-4-chloroproline (Clp). We find that the molecular structure of Ac-Clp-OMe in the solid state is virtually identical to that of Ac-Hyp-OMe. In contrast, the conformational properties of Ac-clp-OMe are similar to those of Ac-Pro-OMe. Ac-Clp-OMe has a stronger preference for a trans amide bond than does Ac-Pro-OMe, whereas Ac-clp-OMe has a weaker preference. (Pro-Clp-Gly)(10) forms triple helices that are significantly more stable than those of (Pro-Pro-Gly)(10). Triple helices of (clp-Pro-Gly)(10) have stability similar to those of (Pro-Pro-Gly)(10). Unlike (Pro-Clp-Gly)(10) and (clp-Pro-Gly)(10), (clp-Clp-Gly)(10) does not form a stable triple helix, presumably due to a deleterious steric interaction between proximal chlorines on different strands. These data, which are consistent with previous work on 4-fluoroprolines and 4-methylprolines, support the importance of stereoelectronic and steric effects in the stability of the collagen triple helix and provide another means to modulate that stability. (     

Neolignans from an Aniba species

The trunk wood of an Amazonian Aniba (Lauraceae) species contains, besides dillapiol and the benzodioxane-type neolignan eusiderin, four bicyclo(3.2.1)octanoid neolignans. These comprise representatives of the canellin-type: the known methoxycanellin-A and the novel compounds characterized as (1R, 3S, 4S, 5S, 6S, 7R)-1-allyl-4-hydroxy-3, 5-dimethoxy-7-methyl-6-(3′-methoxy-4′, 5′-methylenedioxyphenyl)-8-oxo-bicyclo(3.2.1)octane; (1R, 3S, 4S, 5S, 6S, 7R)-1-allyl-4-hydroxy-3, 5-dimethoxy-7-methyl-6-(3′, 4′, 5′-trimethoxyphenyl)-8-oxobicyclo(3.2.1)octane and (1R, 4R, 5R, 6S, 7R, 8S)-1-allyl-4, 8-dihydroxy-5-methoxy-7-methyl-6-(3′-methoxy-4′,5′-methylenedioxyphenyl)-3-oxobicyclo(3.2.1)octane.     

Neolignans from an Aniba species

A benzene extract of the trunk of an Aniba species (Lauraceae) contained benzyl benzoate, benzyl salicylate, sitosterol and the neolignans (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofuran (burchellin); (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,3aR)-3a-allyl-5,7-dimethoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,5S)-5-allyl-5-methoxy-3-methyl-2-veratryl-2,3,5,6-tetrahydro-6-oxo-benzofuran; (2R,3R)-7-methoxy-3-methyl-5-propenyl-2-veratryl-2,3-dihydrobenzofuran; rel-(1R,5R,6R,7R,8S)-1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (guianin); rel-(1S,5S,6S,7R,8R)-1-allyl-8-hydroxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1S,5S,6S,7R,8R)-8-acetoxy-1-allyl-3-hydroxy-5-methoxy-7-methyl-4-oxo-6-piperonyl-bicyclo[3,2,1]oct-2-ene; rel-1S,5S,6S,7R,8R)-8-acetoxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1R,5S,6R,7R)-1-allyl-3-methoxy-7-methyl-4,8-dioxo-6-piperonylbicyclo[3,2,1]oct-2-ene.     

Neolignans from Ocotea catharinensis

The trunk bark of Ocotea catharinensis yielded, besides the known bicyclo(3.2.1)octanoid neolignans canellin-C and 5′-methoxycanellin-C, two epimers rel-(1R,4S and 4R,5S,6R,7S,8R)-1-allyl-4,8-dihydroxy-3,5-dimethoxy-7-methyl-6-piperonyl-bicyclo(3.2.1)oct-2-enes and rel-(1R,5S,6R,7S,8R)-1-allyl-3,8-dihydroxy-5-methoxy-7-methyl-6-piperonyl-4-oxobicyclo(3.2.1)oct-2-ene. The hydrobenzofuranoid neolignans are represented by the equally novel (2S,3S,5R)-5-allyl-5,7-dimethoxy-3-methyl-2-piperonyl-2,3,5,6-tetrahydro-6-oxobenzofuran and (2R,3S,3aS)-3a-allyl-5,7-dimethoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofuran.     

Dolabellane diterpenoids from Aglaia odorata

Dolabellane diterpenoids, (1R,3E,7E,10S,11S,12R)-dolabella-3,7-dien-10,18-diol (1), (1R,3S,7E,11S,12R)-dolabella-4(16),7-dien-3,18-diol (2), (1R,7E,11S,12R)-18-hydroxydolabella-4(16),7-dien-3-one (3), (1R,3S,4S,7E,11S,12R)-3,4-epoxydolabella-7-en-18-ol (4), and (1R,3R,7E,11S,12R)-dolabella-4(16),7,18-trien-3-ol (5), were obtained from the ornamental plant Aglaia odorata. Their structures were characterized on the basis of spectroscopic analyses and further confirmed by X-ray diffraction. Compounds 1 and 5 showed weak cytotoxicity against the human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, and lung cancer A-549 cells.     

Orientation and conformation of lipids in crystals of transmembrane proteins

Orientational order parameters and individual dihedral torsion angles are evaluated for phospholipid and glycolipid molecules that are resolved in X-ray structures of integral transmembrane proteins in crystals. The order parameters of the lipid chains and glycerol backbones in protein crystals are characterised by a much wider distribution of orientational order than is found in fluid lipid bilayers and reconstituted lipid–protein membranes. This indicates that the lipids that are resolved in crystals of membrane proteins are mostly not representative of the entire lipid–protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from C–C bonds in energetically disallowed skew conformations. This suggests configurational heterogeneity of the lipids at a single binding site: eclipsed conformations occur also in the glycerol backbone torsion angles and the C–C torsion angles of the lipid head groups. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1–C2 rotamers found invariably in phospholipid bilayer crystals. Lipid head-group conformations in the protein crystals also do not conform solely to the bent-down conformation, with gauche–gauche configuration of the phosphodiester, that is characteristic of phospholipid bilayer membranes. Stereochemical violations in the protein-bound lipids are evidenced by ester carboxyl groups in non-planar configurations, and even in the cis configuration. Some lipids have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in the phytanyl chains associated with bacteriorhodopsin have the incorrect S configuration.     

1,11-diarylundecan-1-one and 4-aryltetralone neolignans from Virola sebifera

The seed of Virola sebifera contains besides the polyketide 1 - (2′,6′ - dihydroxyphenyl) - 11 - henylundecan - 1 - one, four neolignans: (2S, 3S, 4R) - 4 - hydroxy - 2,3 - dimethyl - 5,6 - methylenedioxy - 4 - piperonyl - 1 - tetralone and its 2-epimer, as well as (2R, 3R, 4S) - 4 - hydroxy - 6,7 - dimethoxy - 2,3 - dimethyl 4 - piperonyl - 1 - tetralone and its (2R, 3S, 4R) - dehydroxy analogue.     

Abietane diterpenoids from Isodon inflexus

Four abietane diterpenoids, inflexanin C, inflexanin D, inflexuside A and inflexuside B, were isolated from the aerial parts of Isodon inflexus. Their respective structures were established by NMR, mass spectrometry and CD as (+)-(1S,4R,5S,7S,8S,10S,13S)-1,7,18-trihydroxy-abieta-9(11)-ene-12-one 1-monoacetate, (+)-(1S,4R,5S,10S,13S)-1,18-dihydroxy-abieta-7,9(11)-diene-12-one 1-monoacetate, (−)-(1S,5S,10S,11R,13R)-1,11,13-trihydroxy-abieta-8-ene-7-one 1-O-β-d-glucopyranoside and (−)-(1S,5S,10S,11R,13R)-1,11,13-trihydroxy-abieta-8-ene-7-one 1-O-(2-O-coumaroyl)-β-d-glucopyranoside. All compounds showed strong inhibitory activity against nitric oxide (NO) production in RAW264.7 lipopolysaccaride (LPS)-activated macrophages.     

Occurrence of juvabione-type and epijuvabione-type sesquiterpenoids in Abies alba

The petrol-soluble fractions from the branchwood of four Abies alba trees were examined. Only two trees contained sufficient amounts of ‘juvabione-type’ insect juvenile hormone analogues for isolation and characterization. The first contained juvabione (4R, 1′R), 4′-dehydrojuvabione (4R, 1′R) and its 4R, 1′S diastereomer in a ratio of 3:1, and juvabiol (4R, 1′R, 3′S), isojuvabiol (4R, 1′R, 3′R) and epijuvabiol (4R, 1′S, 3′S) in an approximate ratio of 7:3:2. 4′-Dehydroepijuvabione (4R, 1′S) was the only ‘juvabione-type’ compound ioslated from the second tree. If it is accepted that juvabione and epijuvabione are enzymatically reduced forms of dehydrojuvabione and dehydroepijuvabione, respectively; then for these two A. alba our results indicate that only one enzyme which is specific for R chirality at C-1′ is present, since epijuvabione is not observed.     

Uniformity,ideality, and hydrogen bonds in transmembrane alpha-helices

Protein environments substantially influence the balance of molecular interactions that generate structural stability. Transmembrane helices exist in the relatively uniform low dielectric interstices of the lipid bilayer, largely devoid of water and with a very hydrophobic distribution of amino acid residues. Here, through an analysis of bacteriorhodopsin crystal structures and the transmembrane helix structure from M2 protein of influenza A, some helices are shown to be exceptionally uniform in hydrogen bond geometry, peptide plane tilt angle, and backbone torsion angles. Evidence from both the x-ray crystal structures and solid-state NMR structure suggests that the intramolecular backbone hydrogen bonds are shorter than their counterparts in water-soluble proteins. Moreover, the geometry is consistent with a dominance of electrostatic versus covalent contributions to these bonds. A comparison of structure as a function of resolution shows that as the structures become better characterized the helices become much more uniform, suggesting that there is a possibility that many more uniform helices will be observed, even among the moderate resolution membrane protein structures that are currently in the Protein Data Bank that do not show such features.     

Structure des caboxines: Alcaloïdes oxindoliques du Cabucala fasciculata

The structures of three new 11-monomethoxy pentacyclic oxindole alkaloids have been elucidated by chemical correlations with reserpinine: caboxine-A was assigned to the allo C₁₉-méthyl α series: 3S, 4R, 7S, 19S; isocaboxine-A and B to the epi-allo C₁₉-methyl α series and have, respectively, the following configurations 3R, 4S, 7S, 19S and 3R, 4S, 7R, 19S.