Helical twists and ß-turns in structures at serine-proline sequences: Stabilization of cis-proline and type VI ß-turns via C-H/O interactions.

Structures at serine-proline sites in proteins were analyzed using a combination of peptide synthesis with structural methods and bioinformatics analysis of the PDB. Dipeptides were synthesized with the proline derivative (2S,4S)-(4-iodophenyl)hydroxyproline [hyp(4-I-Ph)]. The crystal structure of Boc-Ser-hyp(4-I-Ph)-OMe had two molecules in the unit cell. One molecule exhibited cis-proline and a type VIa2 ß-turn (BcisD). The cis-proline conformation was stabilized by a C-H/O interaction between Pro C-Hα and the Ser side-chain oxygen. NMR data were consistent with stabilization of cis-proline by a C-H/O interaction in solution. The other crystallographically observed molecule had trans-Pro and both residues in the PPII conformation. Two conformations were observed in the crystal structure of Ac-Ser-hyp(4-I-Ph)-OMe, with Ser adopting PPII in one and the ß conformation in the other, each with Pro in the δ conformation and trans-Pro. Structures at Ser-Pro sequences were further examined via bioinformatics analysis of the PDB and via DFT calculations. Ser-Pro versus Ala-Pro sequences were compared to identify bases for Ser stabilization of local structures. C-H/O interactions between the Ser side-chain Oγ and Pro C-Hα were observed in 45% of structures with Ser-cis-Pro in the PDB, with nearly all Ser-cis-Pro structures adopting a type VI ß-turn. 53% of Ser-trans-Pro sequences exhibited main-chain COi•••HNi+3 or COi•••HNi+4 hydrogen bonds, with Ser as the i residue and Pro as the i + 1 residue. These structures were overwhelmingly either type I ß-turns or N-terminal capping motifs on α-helices or 310-helices. These results indicate that Ser-Pro sequences are particularly potent in favoring these structures. In each, Ser is in either the PPII or ß conformation, with the Ser Oγ capable of engaging in a hydrogen bond with the amide N-H of the i + 2 (type I ß-turn or 310-helix; Ser χ1 t) or i + 3 (α-helix; Ser χ1 g+) residue. Non-proline cis amide bonds can also be stabilized by C-H/O interactions.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Acyl Capping Group Identity Effects on α-Helicity: On the Importance of Amide·Water Hydrogen Bonds to α-Helix Stability.

Acyl capping groups stabilize α-helices relative to free N-termini by providing one additional C═Oi···Hi+4-N hydrogen bond. The electronic properties of acyl capping groups might also directly modulate α-helix stability: electron-rich N-terminal acyl groups could stabilize the α-helix by strengthening both i/i + 4 hydrogen bonds and i/i + 1 n → π* interactions. This hypothesis was tested in peptides X-AKAAAAKAAAAKAAGY-NH2, where X = different acyl groups. Surprisingly, the most electron-rich acyl groups (pivaloyl and iso-butyryl) strongly destabilized the α-helix. Moreover, the formyl group induced nearly identical α-helicity to that of the acetyl group, despite being a weaker electron donor for hydrogen bonds and for n → π* interactions. Other acyl groups exhibited intermediate α-helicity. These results indicate that the electronic properties of the acyl carbonyl do not directly determine the α-helicity in peptides in water. In order to understand these effects, DFT calculations were conducted on α-helical peptides. Using implicit solvation, α-helix stability correlated with acyl group electronics, with the pivaloyl group exhibiting closer hydrogen bonds and n → π* interactions, in contrast to the experimental results. However, DFT and MD calculations with explicit water solvation revealed that hydrogen bonding to water was impacted by the sterics of the acyl capping group. Formyl capping groups exhibited the closest water-amide hydrogen bonds, while pivaloyl groups exhibited the longest. In α-helices in the PDB, the highest frequency of close amide-water hydrogen bonds is observed when the N-cap residue is Gly. The combination of experimental and computational results indicates that solvation (hydrogen bonding of water) to the N-terminal amide groups is a central determinant of α-helix stability.

N-Terminal Proline Editing for the Synthesis of Peptides with Mercaptoproline and Selenoproline: Mechanistic Insights Lead to Greater Efficiency in Proline Native Chemical Ligation.

Native chemical ligation (NCL) at proline has been limited by cost and synthetic access. In addition, prior examples of NCL using mercaptoproline have exhibited stalling of the reaction after thioester exchange, due to inefficient S → N acyl transfer. Herein, we develop methods, using inexpensive Boc-4R-hydroxyproline, for the solid-phase synthesis of peptides containing N-terminal 4R-mercaptoproline and 4R-selenoproline. The synthesis proceeds via proline editing on the N-terminus of fully synthesized peptides on the solid phase, converting an N-terminal Boc-4R-hydroxyproline to the 4S-bromoproline, followed by an SN2 reaction with potassium thioacetate or selenobenzoic acid. After cleavage from the resin and deprotection, peptides with functionalized N-terminal proline amino acids were obtained. NCL reactions with mercaptoproline proceeded slowly under standard NCL conditions, with the S-acyl transthioesterification intermediate observed as a major species. Computational investigations indicated that the bicyclic intermediates and transition states for S → N acyl transfer are sufficiently low in energy (10-15 kcal mol-1 above starting material) that ring strain cannot explain the slow S → N acyl transfer. Instead, the bicyclic zwitterionic tetrahedral intermediate has a low barrier for reversion to the S-acyl intermediate, causing reversion to the thioester (reverse reaction) to occur preferentially over elimination to generate the amide (forward reaction). We hypothesized that a buffer capable of general acid and/or general base catalysis could promote S → N acyl transfer and thus achieve greater efficiency in proline NCL. In the presence of 2 M imidazole at pH 6.8, NCL with mercaptoproline proceeded efficiently to generate the peptide with a native amide bond. NCL with selenoproline also proceeded efficiently to generate the desired products when a thiophenol thioester was employed as a ligation partner. After desulfurization or deselenization, the products obtained were identical to those synthesized directly, confirming that the solid-phase proline editing reactions proceeded stereospecifically and without epimerization.

An Inherent Difference between Serine and Threonine Phosphorylation: Phosphothreonine Strongly Prefers a Highly Ordered, Compact, Cyclic Conformation.

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted ϕ (ϕ ∼ -60°) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n → π* interaction between consecutive carbonyls, and an n → σ* interaction between the phosphate Oγ lone pair and the antibonding orbital of C-Hß that restricts the χ2 side-chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic proline's backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are ϕ,ψ = polyproline II helix > α-helix (ϕ ∼ -60°); χ1 = g-; χ2 ∼ +115° (eclipsed C-H/O-P bonds). This structural signature is observed in diverse proteins, including in the activation loops of protein kinases and in protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

Synthesis and conformational preferences of peptides and proteins with cysteine sulfonic acid.

Cysteine sulfonic acid (Cys-SO3H; cysteic acid) is an oxidative post-translational modification of cysteine, resulting from further oxidation from cysteine sulfinic acid (Cys-SO2H). Cysteine sulfonic acid is considered an irreversible post-translational modification, which serves as a biomarker of oxidative stress that has resulted in oxidative damage to proteins. Cysteine sulfonic acid is anionic, as a sulfonate (Cys-SO3-; cysteate), in the ionization state that is almost exclusively present at physiological pH (pKa ∼ -2). In order to understand protein structural changes that can occur upon oxidation to cysteine sulfonic acid, we analyzed its conformational preferences, using experimental methods, bioinformatics, and DFT-based computational analysis. Cysteine sulfonic acid was incorporated into model peptides for α-helix and polyproline II helix (PPII). Within peptides, oxidation of cysteine to the sulfonic acid proceeds rapidly and efficiently at room temperature in solution with methyltrioxorhenium (MeReO3) and H2O2. Peptides containing cysteine sulfonic acid were also generated on solid phase using trityl-protected cysteine and oxidation with MeReO3 and H2O2. Using methoxybenzyl (Mob)-protected cysteine, solid-phase oxidation with MeReO3 and H2O2 generated the Mob sulfone precursor to Cys-SO2- within fully synthesized peptides. These two solid-phase methods allow the synthesis of peptides containing either Cys-SO3- or Cys-SO2- in a practical manner, with no solution-phase synthesis required. Cys-SO3- had low PPII propensity for PPII propagation, despite promoting a relatively compact conformation in ϕ. In contrast, in a PPII initiation model system, Cys-SO3- promoted PPII relative to neutral Cys, with PPII initiation similar to Cys thiolate but less than Cys-SO2- or Ala. In an α-helix model system, Cys-SO3- promoted α-helix near the N-terminus, due to favorable helix dipole interactions and favorable α-helix capping via a sulfonate-amide side chain-main chain hydrogen bond. Across all peptides, the sulfonate side chain was significantly less ordered than that of the sulfinate. Analysis of Cys-SO3- in the PDB revealed a very strong propensity for local (i/i or i/i + 1) side chain-main chain sulfonate-amide hydrogen bonds for Cys-SO3-, with >80% of Cys-SO3- residues exhibiting these interactions. DFT calculations conducted to explore these conformational preferences indicated that side chain-main chain hydrogen bonds of the sulfonate with the intraresidue amide and/or with the i + 1 amide were favorable. However, hydrogen bonds to water or to amides, as well as interactions with oxophilic metals, were weaker for the sulfonate than the sulfinate, due to lower charge density on the oxygens in the sulfonate.

Proline C-H Bonds as Loci for Proline Assembly via C-H/O Interactions.

Proline residues within proteins lack a traditional hydrogen bond donor. However, the hydrogens of the proline ring are all sterically accessible, with polarized C-H bonds at Hα and Hδ that exhibit greater partial positive character and can be utilized as alternative sites for molecular recognition. C-H/O interactions, between proline C-H bonds and oxygen lone pairs, have been previously identified as modes of recognition within protein structures and for higher-order assembly of protein structures. In order to better understand intermolecular recognition of proline residues, a series of proline derivatives was synthesized, including 4R-hydroxyproline nitrobenzoate methyl ester, acylated on the proline nitrogen with bromoacetyl and glycolyl groups, and Boc-4S-(4-iodophenyl)hydroxyproline methyl amide. All three derivatives exhibited multiple close intermolecular C-H/O interactions in the crystallographic state, with H⋅⋅⋅O distances as close as 2.3 Å. These observed distances are well below the 2.72 Šsum of the van der Waals radii of H and O, and suggest that these interactions are particularly favorable. In order to generalize these results, we further analyzed the role of C-H/O interactions in all previously crystallized derivatives of these amino acids, and found that all 26 structures exhibited close intermolecular C-H/O interactions. Finally, we analyzed all proline residues in the Cambridge Structural Database of small-molecule crystal structures. We found that the majority of these structures exhibited intermolecular C-H/O interactions at proline C-H bonds, suggesting that C-H/O interactions are an inherent and important mode for recognition of and higher-order assembly at proline residues. Due to steric accessibility and multiple polarized C-H bonds, proline residues are uniquely positioned as sites for binding and recognition via C-H/O interactions.

Solvation stabilizes intercarbonyl n→π* interactions and polyproline II helix.

n→π* interactions between consecutive carbonyls stabilize the α-helix and polyproline II helix (PPII) conformations in proteins. n→π* interactions have been suggested to provide significant conformational biases to the disordered states of proteins. To understand the roles of solvation on the strength of n→π* interactions, computational investigations were conducted on a model n→π* interaction, the twisted-parallel-offset formaldehyde dimer, as a function of explicit solvation of the donor and acceptor carbonyls, using water and HF. In addition, the effects of urea, thiourea, guanidinium, and monovalent cations on n→π* interaction strength were examined. Solvation of the acceptor carbonyl significantly strengthens the n→π* interaction, while solvation of the donor carbonyl only modestly weakens the n→π* interaction. The n→π* interaction strength was maximized with two solvent molecules on the acceptor carbonyl. Urea stabilized the n→π* interaction via simultaneous engagement of both oxygen lone pairs on the acceptor carbonyl. Solvent effects were further investigated in the model peptides Ac-Pro-NMe2, Ac-Ala-NMe2, and Ac-Pro2-NMe2. Solvent effects in peptides were similar to those in the formaldehyde dimer, with solvation of the acceptor carbonyl increasing n→π* interaction strength and resulting in more compact conformations, in both the proline endo and exo ring puckers, as well as a reduction in the energy difference between these ring puckers. Carbonyl solvation leads to an energetic preference for PPII over both the α-helix and ß/extended conformations, consistent with experimental data that protic solvents and protein denaturants both promote PPII. Solvation of the acceptor carbonyl weakens the intraresidue C5 hydrogen bond that stabilizes the ß conformation.

Perfluoro-tert-Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by 19F NMR.

19F NMR spectroscopy provides the ability to quantitatively analyze single species in complex solutions but is often limited by the modest sensitivity inherent to NMR. 4R- and 4S-Perfluoro-tert-buyl hydroxyproline contain 9 equivalent fluorines, in amino acids with strong conformational preferences. In order to test the ability to use these amino acids as sensitive probes of protein modifications, the perfluoro-tert-buyl hydroxyprolines were incorporated into substrate peptides of the protein kinases PKA and Akt. Peptides containing each diastereomeric proline were rapidly phosphorylated by each protein kinase and exhibited 19F chemical shift changes as a result of phosphorylation. The sensitivity of the perfluoro-tert-butyl group allowed quantitative analysis of the kinetics of phosphorylation over three half-lives at single-digit micromolar concentrations of each species. The distinct conformational preferences of these amino acids allowed the optimization of the substrate with a conformationally matched amino acid, in order to maximize the rate of phosphorylation. PKA preferred the 4R-amino acid at the -1 position, whereas the closely related AGC kinase Akt preferred the 4S-amino acid. These data, combined with analysis of structures of the Michaelis complexes of these kinases in the PDB, suggest that PKA recognizes the PPII conformation at the P-1 position relative to the phosphorylation site, while Akt/PKB recognizes an extended conformation at this position. These results suggest that conformational targeting may be employed to increase specificity in recognition by protein kinases. Perfluoro-tert-butyl hydroxyprolines were applied to the real-time detection and quantification of PKA activity and inhibition of PKA activity in HeLa cell extracts via 19F NMR spectroscopy. The coupling of proline ring pucker with main chain conformation suggests broad application of perfluoro-tert-butyl hydroxyprolines in molecular sensing and imaging.

Cysteine oxidation to the sulfinic acid induces oxoform-specific lanthanide binding and fluorescence in a designed peptide.

Cysteine sulfinic acid (Cys-SO2-) is a protein post-translational modification that is formed reversibly under oxidative conditions. A short, encodable peptide was developed whose metal binding and terbium luminescence are dependent on cysteine (Cys) oxidation to the sulfinic acid. The protein design is based on the modification of a key metal-binding aspartate (Asp) in a canonical EF-Hand motif (DKDADGWISPAEAK) to Cys. In this design, Cys in the thiol oxidation state does not mimic the native Asp, and thus the peptide binds terbium(III) (Tb3+) poorly and exhibits weak terbium luminescence (fluorescence). In contrast, when Cys is oxidized to the Cys sulfinic acid oxoform, the Cys sulfinate effectively mimics Asp, resulting in a significant increase in terbium affinity and luminescence. Asp residues at positions 1, 3, and 5 of the EF-Hand motif were examined as potential sites for Cys oxidation-responsive metal binding. The peptide with Cys at residue 1 exhibited the highest Tb3+ affinity in both oxidation states. The peptide with Cys at residue 3 exhibited a 4.2-fold distinction in affinity between the oxidation states. Most significantly, the peptide with Cys at residue 5 had only modest Tb3+ affinity as the Cys thiol, but exhibited a 30-fold increase in Tb3+ affinity and an 18-fold increase in Tb3+ luminescence on Cys oxidation to the sulfinic acid. This peptide (Ac-DKDACGWISPAEAK-NH2) exhibited selective Tb3+ binding via Cys-SO2- over the thiol, S-glutathionyl, S-nitrosyl, and sulfonic acid oxoforms, indicating substantially greater Lewis basicity of the sulfinate than the sulfonate. NMR spectroscopy and quantum homology modeling indicated that the designed peptide binds metal with an overall geometry similar to that of an EF-Hand motif, with the Cys sulfinate effectively replacing Asp as a metal-binding ligand. This peptide was applied to detect Cys oxidation to the sulfinic acid by fluorescence spectroscopy, suggesting its broader application in understanding Cys sulfinic acid biology.

Structural preferences of cysteine sulfinic acid: The sulfinate engages in multiple local interactions with the peptide backbone.

Cysteine sulfinic acid (Cys-SO2-) is a non-enzymatic oxidative post-translational modification (PTM) that has been identified in hundreds of proteins. However, the effects of cysteine sulfination are in most cases poorly understood. Cys-SO2- is structurally distinctive, with long sulfur-carbon and sulfur-oxygen bonds, and with tetrahedral geometry around sulfur due to its lone pair. Cys-SO2- thus has a unique range of potential interactions with the protein backbone which could facilitate protein structural changes. Herein, the structural effects of cysteine oxidation to the sulfinic acid were investigated in model peptides and folded proteins using NMR spectroscopy, circular dichroism, bioinformatics, and computational studies. In the PDB, Cys-SO2- shows a greater preference for α-helix than Cys. In addition, Cys-SO2- is more commonly found in structures with φ > 0, including in multiple types of ß-turn. Sulfinate oxygens engage in hydrogen bonds with adjacent (i or i + 1) amide hydrogens. Over half of sulfinates have at least one hydrogen bond with an adjacent amide, and several structures have hydrogen bonds with both adjacent amides. Alternately, sulfur or either oxygen can act as an electron donor for n→π* interactions with the backbone carbonyl of the same residue, as indicated by frequent S⋯CO or O⋯CO distances below the sums of their van der Waals radii in protein structures. In peptides, Cys-SO2- favored α-helical structure at the N-terminus, consistent with helix dipole effects and backbone hydrogen bonds with the sulfinate promoting α-helix. Cys-SO2- has only modestly greater polyproline II helix propensity than Cys-SH, likely due to competition from multiple side chain-backbone interactions. Cys-SO2- stabilizes the i+1 position of a ß-turn relative to Cys-SH. Within proteins, the range of side chain-main chain interactions available to Cys-SO2- compared to Cys-SH provides a basis for potential changes in protein structure and function due to cysteine oxidation to the sulfinic acid.

SAR by 1D NMR.

1D NMR spectroscopy is a standard technique in the characterization of organic molecules. 1D NMR data inherently provide information on the conformational preferences of molecules, but this information is typically overlooked beyond the determination of compound identity and purity. Balazs and co-workers describe the use of routine 1D NMR spectra (chemical shifts, chemical shift dispersion, coupling constants) of molecules free in solution, in combination with protein target binding data, in order to identify conformational signatures of molecules when bound to their targets. Via case studies, they demonstrate the application of these conformational signatures observed in simple 1D NMR spectra in the optimization of compounds for medicinal chemistry, with particular application to the development of optimized linkers in the synthesis of macrocycles.

The Distinct Conformational Landscapes of 4S-Substituted Prolines That Promote an endo Ring Pucker.

4-Substitution on proline directly impacts protein main chain conformational preferences. The structural effects of N-acyl substitution and of 4-substitution were examined by NMR spectroscopy and X-ray crystallography on minimal molecules with a proline 4S-nitrobenzoate. The effects of N-acyl substitution on conformation were attenuated in the 4S-nitrobenzoate context, due to the minimal role of the n→π* interaction in stabilizing extended conformations. By X-ray crystallography, an extended conformation was observed for most molecules. The formyl derivative adopted a δ conformation that is observed at the i+2 position of ß-turns. Computational analysis indicated that the structures observed crystallographically represent the inherent conformational preferences of 4S-substituted prolines with electron-withdrawing 4-position substituents. The divergent conformational preferences of 4R- and 4S-substituted prolines suggest their wider structure-specific application in molecular design. In particular, the proline endo ring pucker favored by 4S-substituted prolines uniquely promotes the δ conformation [(ϕ, ψ) ≈(-80°, 0°)] found in ß-turns. In contrast to other acyl capping groups, the pivaloyl group strongly promoted trans amide bond and polyproline II helix conformation, with a close n→π* interaction in the crystalline state, despite the endo ring pucker, suggesting its special capabilities in promoting compact conformations in ϕ due to its strongly electron-donating character.

Design of a Protein Motif Responsive to Tyrosine Nitration and an Encoded Turn-Off Sensor of Tyrosine Nitration.

Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-hand metal-binding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. Nuclear magnetic resonance spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. To develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF-hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with a tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity ( Kd = 1.6 µM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched upon nitration. The greater terbium affinity upon tyrosine nitration resulted in a large dynamic range and sensitivity to substoichiometric nitration. An improved approach for the synthesis of peptides containing nitrotyrosine was also developed, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.

Phosphorylation-dependent protein design: design of a minimal protein kinase-inducible domain.

Protein kinases and phosphatases modulate protein structure and function, which in turn regulate cellular activities. The development of novel proteins and protein motifs that are responsive to protein phosphorylation provides new ways to probe the functions of individual protein kinases and the intracellular effects of their activation and downregulation. Herein we develop a minimal motif that is responsive to protein phosphorylation, termed a minimal protein kinase-inducible domain. The encodable protein motif comprises a 7- or 8-residue sequence (DKDADXW or DKDADXXW), derived from EF-Hand calcium-binding domains, that is necessary but not sufficient for binding terbium, combined with a protein phosphorylation site (Ser or Thr at residue 9) that, upon phosphorylation, completes the metal-binding motif. Thus, the motif binds metal poorly and exhibits weak terbium luminescence when not phosphorylated. Upon phosphorylation, the peptide binds metal with significantly higher affinity and exhibits robust terbium luminescence. Phosphorylation results in up to a 23× increase in terbium luminescence. Minimal phosphorylation-dependent motifs as small as 9 residues (DKDADGWIS) were developed. NMR spectroscopy on this lanthanum(iii)·phosphopeptide complex confirmed that binding occurs in a manner similar to that in an EF-Hand, despite the absence of the conserved Glu12 typically present in an EF-Hand. By combining molecular design with known protein kinase recognition sequences, minimal protein kinase-inducible domains were developed that were responsive to phosphorylation by Protein Kinase A (PKA: DKDADRRW(S/pS)IIAK), Protein Kinase C (PKC: DKDADGWI(T/pT)FRRKA), and Casein Kinase 1 (CK1: DKDADDWA(S/pS)I). Phosphorylation by PKA was quantified in HeLa cell extracts, with a 4.4× increase in fluorescence (terbium luminescence) observed at 544 nm. The optimized minimal motif includes alternating aspartate residues at positions 1, 3, and 5, plus binding through the main-chain carbonyl at position 7; a lysine at position 2 to provide electrostatic balance and reduce binding in the absence of phosphorylation; an alanine at residue 4 to promote the αL conformation observed at that position of the EF Hand; a tryptophan at residue 7 or 8 to sensitize terbium luminescence; and a phosphorylation site with serine or threonine at residue 9. Residues at positions 6; 7 or 8; and 10 or later may be changed to provide kinase specificity. In the CK1-responsive peptide, the acidic residues in the proto-terbium-binding motif are employed as part of the kinase recognition sequence. This work thus presents fundamental rules for the design of compact phosphorylation-responsive terbium-binding motifs, with potential further application to motifs responsive to other protein post-translational modifications.

Electronic and Steric Control of n→π* Interactions: Stabilization of the α-Helix Conformation without a Hydrogen Bond.

The preferred conformations of peptides and proteins are dependent on local interactions that bias the conformational ensemble. The n→π* interaction between consecutive carbonyls promotes compact conformations, including the α-helix and polyproline II helix. In order to further understand the n→π* interaction and to develop methods to promote defined conformational preferences through acyl N-capping motifs, a series of peptides was synthesized in which the electronic and steric properties of the acyl group were modified. Using NMR spectroscopy, van't Hoff analysis of enthalpies, X-ray crystallography, and computational investigations, we observed that more electron-rich donor carbonyls (pivaloyl, iso-butyryl, propionyl) promote stronger n→π* interactions and more compact conformations than acetyl or less electron-rich donor carbonyls (methoxyacetyl, fluoroacetyl, formyl). X-ray crystallography indicates a strong, electronically tunable preference for the α-helix conformation, as observed directly on the φ and ψ torsion angles. Electron-donating acyl groups promote the α-helical conformation, even in the absence of the hydrogen bonding that stabilizes the α-helix. In contrast, electron-withdrawing acyl groups led to more extended conformations. More sterically demanding groups can promote trans amide bonds independent of the electronic effect on n→π* interactions. Chloroacetyl groups additionally promote n→π* interactions through the interaction of the chlorine lone pair with the proximal carbonyl π*. These data provide additional support for an important role of n→π* interactions in the conformational ensemble of disordered or unfolded proteins. Moreover, this work suggests that readily incorporated acyl N-capping motifs that modulate n→π* interactions may be employed rationally to promote conformational biases in peptides, with potential applications in molecular design and medicinal chemistry.

Redox-Responsive Protein Design: Design of a Small Protein Motif Dependent on Glutathionylation.

Cysteine S-glutathionylation is a protein post-translational modification that promotes cellular responses to changes in oxidative conditions. The design of protein motifs that directly depend on defined changes to protein side chains provides new methods for probing diverse protein post-translational modifications. A canonical, 12-residue EF-hand motif was redesigned to be responsive to cysteine glutathionylation. The key design principle was the replacement of the metal-binding Glu12 carboxylate of an EF-hand with a motif capable of metal binding via a free carboxylate in the glutathione-conjugated peptide. In the optimized peptide (DKDADGWCG), metal binding and terbium luminescence were dependent on glutathionylation, with weaker metal binding in the presence of reduced cysteine but increased metal affinity and a 3.5-fold increase in terbium luminescence at 544 nm when cysteine was glutathionylated. Nuclear magnetic resonance spectroscopy indicated that the structure at all residues of the glutathionylated peptide changed in the presence of metal, with chemical shift changes consistent with the adoption of an EF-hand-like structure in the metal-bound glutathionylated peptide. This small protein motif consists of canonical amino acids and is thus genetically encodable, for its potential use as a localized tag to probe protein glutathionylation.

Perfluoro-tert-butyl Homoserine Is a Helix-Promoting, Highly Fluorinated, NMR-Sensitive Aliphatic Amino Acid: Detection of the Estrogen Receptor·Coactivator Protein-Protein Interaction by 19F NMR.

Highly fluorinated amino acids can stabilize proteins and complexes with proteins, via enhanced hydrophobicity, and provide novel methods for identification of specific molecular events in complex solutions, via selective detection by 19F NMR and the absence of native 19F signals in biological contexts. However, the potential applications of 19F NMR in probing biological processes are limited both by the strong propensities of most highly fluorinated amino acids for the extended conformation and by the relatively modest sensitivity of NMR spectroscopy, which typically constrains measurements to mid-micromolar concentrations. Herein, we demonstrate that perfluoro-tert-butyl homoserine exhibits a propensity for compact conformations, including α-helix and polyproline helix (PPII), that is similar to that of methionine. Perfluoro-tert-butyl homoserine has nine equivalent fluorines that do not couple to any other nuclei, resulting in a sharp singlet that can be sensitively detected rapidly at low micromolar concentrations. Perfluoro-tert-butyl homoserine was incorporated at sites of leucine residues within the α-helical LXXLL short linear motif of estrogen receptor (ER) coactivator peptides. A peptide containing perfluoro-tert-butyl homoserine at position i + 3 of the ER coactivator LXXLL motif exhibited a Kd of 2.2 µM for the estradiol-bound estrogen receptor, similar to that of the native ligand. 19F NMR spectroscopy demonstrated the sensitive detection (5 µM concentration, 128 scans) of binding of the peptide to the ER and of inhibition of protein-protein interaction by the native ligand or by the ER antagonist tamoxifen. These results suggest diverse potential applications of perfluoro-tert-butyl homoserine in probing protein function and protein-protein interfaces in complex solutions.

Insights into Thiol-Aromatic Interactions: A Stereoelectronic Basis for S-H/π Interactions.

Thiols can engage favorably with aromatic rings in S-H/π interactions, within abiological systems and within proteins. However, the underlying bases for S-H/π interactions are not well understood. The crystal structure of Boc-l-4-thiolphenylalanine tert-butyl ester revealed crystal organization centered on the interaction of the thiol S-H with the aromatic ring of an adjacent molecule, with a through-space Hthiol···Caromatic distance of 2.71 Å, below the 2.90 Å sum of the van der Waals radii of H and C. The nature of this interaction was further examined by DFT calculations, IR spectroscopy, solid-state NMR spectroscopy, and analysis of the Cambridge Structural Database. The S-H/π interaction was found to be driven significantly by favorable molecular orbital interactions, between an aromatic π donor orbital and the S-H σ* acceptor orbital (a π → σ* interaction). For comparison, a structural analysis of O-H/π interactions and of cation/π interactions of alkali metal cations with aromatic rings was conducted. Na+ and K+ exhibit a significant preference for the centroid of the aromatic ring and distances near the sum of the van der Waals and ionic radii, as expected for predominantly electrostatic interactions. Li+ deviates substantially from Na+ and K+. The S-H/π interaction differs from classical cation/π interactions by the preferential alignment of the S-H σ* toward the ring carbons and an aromatic π orbital rather than toward the aromatic centroid. These results describe a potentially broadly applicable approach to understanding the interactions of weakly polar bonds with π systems.

Synthesis of Perfluoro-tert-butyl Tyrosine, for Application in 19F NMR, via a Diazonium-Coupling Reaction.

A practical synthesis of the novel highly fluorinated amino acid Fmoc-perfluoro-tert-butyl tyrosine was developed. The sequence proceeds in two steps from commercially available Fmoc-4-NH2-phenylalanine via diazotization followed by diazonium coupling reaction with perfluoro-tert-butanol. In peptides, perfluoro-tert-butyl tyrosine was detected in 30 s by NMR spectroscopy at 500 nM peptide concentration due to nine chemically equivalent fluorines that are a sharp singlet by 19F NMR. Perfluoro-tert-butyl ether has an estimated σp Hammett substituent constant of +0.30.