Determination of the structures of symmetric protein oligomers from NMR chemical shifts and residual dipolar couplings

Symmetric protein dimers, trimers, and higher-order cyclic oligomers play key roles in many biological processes. However, structural studies of oligomeric systems by solution NMR can be difficult due to slow tumbling of the system and the difficulty in identifying NOE interactions across protein interfaces. Here, we present an automated method (RosettaOligomers) for determining the solution structures of oligomeric systems using only chemical shifts, sparse NOEs, and domain orientation restraints from residual dipolar couplings (RDCs) without a need for a previously determined structure of the monomeric subunit. The method integrates previously developed Rosetta protocols for solving the structures of monomeric proteins using sparse NMR data and for predicting the structures of both nonintertwined and intertwined symmetric oligomers. We illustrated the performance of the method using a benchmark set of nine protein dimers, one trimer, and one tetramer with available experimental data and various interface topologies. The final converged structures are found to be in good agreement with both experimental data and previously published high-resolution structures. The new approach is more readily applicable to large oligomeric systems than conventional structure-determination protocols, which often require a large number of NOEs, and will likely become increasingly relevant as more high-molecular weight systems are studied by NMR.     

Structural and spectroscopic studies of peptoid oligomers with alpha-chiral aliphatic side chains

Substantial progress has been made in the synthesis and characterization of various oligomeric molecules capable of autonomous folding to well-defined, repetitive secondary structures. It is now possible to investigate sequence-structure relationships and the driving forces for folding in these systems. Here, we present detailed analysis by X-ray crystallography, NMR, and circular dichroism (CD) of the helical structures formed by N-substituted glycine (or "peptoid") oligomers with alpha-chiral, aliphatic side chains. The X-ray crystal structure of a N-(1-cyclohexylethyl)glycine pentamer, the first reported for any peptoid, shows a helix with cis-amide bonds, approximately 3 residues per turn, and a pitch of approximately 6.7 A. The backbone dihedral angles of this pentamer are similar to those of a polyproline type I peptide helix, in agreement with prior modeling predictions. This crystal structure likely represents the major solution conformers, since the CD spectra of analogous peptoid hexamers, dodecamers, and pentadecamers, composed entirely of either (S)-N-(1-cyclohexylethyl)glycine or (S)-N-(sec-butyl)glycine monomers, also have features similar to those of the polyproline type I helix. Furthermore, this crystal structure is similar to a solution NMR structure previously described for a peptoid pentamer comprised of chiral, aromatic side chains, which suggests that peptoids containing either aromatic or aliphatic alpha-chiral side chains adopt fundamentally similar helical structures in solution, despite distinct CD spectra. The elucidation of detailed structural information for peptoid helices with alpha-chiral aliphatic side chains will facilitate the mimicry of biomolecules, such as transmembrane protein domains, in a distinctly stable form.     

Evidence of secondary structure by high-resolution magic angle spinning NMR spectroscopy of a bioactive peptide bound to different solid supports.

The structure of the 19-amino acid peptide epitope, corresponding to the 141-159 sequence of capsid viral protein VP1 of foot-and-mouth disease virus (FMDV), bound to three different resins, namely, polystyrene-MBHA, PEGA, and POEPOP, has been determined by high-resolution magic angle spinning (HRMAS) NMR spectroscopy. A combination of homonuclear and heteronuclear bidimensional experiments was used for the complete peptide resonance assignment and the qualitative characterization of the peptide folding. The influence of the chemicophysical nature of the different polymers on the secondary structure of the covalently attached FMDV peptide was studied in detail. In the case of polystyrene-MBHA and polyacrylamide-PEGA resins, the analysis of the 2D spectra was hampered by missing signals and extensive overlaps, and only a propensity toward a peptide secondary structure could be derived from the assigned NOE correlations. When the FMDV peptide was linked to the polyoxyethylene-based POEPOP resin, it was found to adopt in dimethylformamide a helical conformation encompassing the C-terminal domain from residues 152 to 159. This conformation is very close to that of the free peptide previously analyzed in 2,2,2-trifluoroethanol. Our study clearly demonstrates that a regular helical structure can be adopted by a resin-bound bioactive peptide. Moreover, a change in the folding was observed when the same peptide-POEPOP conjugate was swollen in aqueous solution, displaying the same conformational features as the free peptide in water. The possibility of studying solid-supported ordered secondary structures by the HRMAS NMR technique in a wide range of solvents can be extended either to other biologically relevant peptides and proteins or to new synthetic oligomers.     

(¹⁵N ± ¹³C') edited (4, 3)D-H(CC)CONH TOCSY and (4, 3)D-NOESY HNCO experiments for unambiguous side chain and NOE assignments of proteins with high shift degeneracy

Well-resolved and unambiguous through-bond correlations and NOE data are crucial for high-quality protein structure determination by NMR. In this context, we present here (4, 3)D reduced dimensionality (RD) experiments: H(CC)CONH TOCSY and NOESY HNCO--which instead of (15)N shifts exploit the linear combination of (15)N(i) and (13)C'(i-1) shifts (where i is a residue number) to resolve the through-bond (1)H-(1)H correlations and through-space (1)H-(1)H NOEs. The strategy makes use of the fact that (15)N and (13)C' chemical shifts when combined linearly provide a dispersion which is better compared to those of the individual chemical shifts. The extended dispersion thus available in these experiments will help to obtain the unambiguous side chain and accurate NOE assignments especially for medium-sized alpha-helical or partially unstructured proteins [molecular weight (MW) between 12-15 kDa] as well as higher MW (between 15-25 kDa) folded proteins where spectral overlap renders inaccurate and ambiguous NOEs. Further, these reduced dimensionality experiments in combination with routinely used (15)N and (13)C' edited TOCSY and NOESY experiments will provide an alternative way for high-quality NMR structure determination of large unstable proteins (with very high shift degeneracy), which are not at all amenable to 4D NMR. The utility of these experiments has been demonstrated here using (13)C/(15)N labeled ubiquitin (76 aa) protein.     

Design,synthesis, and NMR structure of linear and cyclic oligomers containing novel furanoid sugar amino acids

Sugar Amino Acids (SAAs) are sugar moieties containing at least one amino and one carboxyl group. The straightforward synthesis of two furanoid SAAs, 3-amino-3-deoxy-1,2-isopropylidene-alpha-D-ribofuranoic acid (f-SAA1) and 3-amino-3-deoxy-1,2-isopropylidene-alpha-D-allofuranoic acid (f-SAA2) starting from diacetone glucose, is described. These SAAs were used as structural templates aiming at new structures for peptidomimetic drug design. f-SAA1 resembles a beta-amino acid, whereas f-SAA2 is a gamma-amino acid mimetic. Thus, for the synthesis of the mixed, linear and cyclic oligomers of f-SAA1, beta-homo-glycine (beta-hGly, also called beta-alanine) was chosen as an amino acid counterpart, while for the oligomer of f-SAA2 gamma-amino butyric acid (GABA) was chosen. Fmoc-[f-SAA1-beta-hGly](3)-OH (3) and cyclo[f-SAA1-beta-hGly](3) (5) resemble linear and cyclic beta-peptides with a very different substitution pattern, compared with the beta-peptides known so far in the literature, whereas Fmoc-[f-SAA2-GABA](3)-OH (4) resembles a gamma-peptide. The linear f-SAA oligomers 3 and 4 were synthesized on the solid-phase using Fmoc strategy. 23 unambiguous interresidue NOE contacts (from a total of 76 NOE values), obtained from extensive NMR studies in C(3)CN, were used in subsequent simulated annealing and MD calculations, to elucidate the 12/10/12-helical structure of oligomer 3 in CH(3)CN. The results indicate that f-SAA1 strongly induces a secondary structure. A characteristic CD curve for the linear oligomer 3 is observed up to 75 degrees C in both CH(3)CN and CH(3)CN/H(2)O, even though 3 contains beta-hGly, which is known to destabilize helices. By contrast, 4 does not seem to form a stable conformation in solution. The cyclic SAA containing oligomer cyclo [f-SAA1-beta-hGly](3) (5) exhibits a C(3) symmetric conformation on the NMR chemical shift time scale.     

Spin system assignment of homo-o-phenylene ethynylene oligomers

We previously reported the synthesis and solution characterization of short o-phenylene ethynylene (oPE) foldamers. Proton correlation techniques are not adequate for NMR assignment in these compounds as the ethynylene linkers interrupt proton connectivity. In order to facilitate structural characterization and more fully harness the power of NMR, it is necessary to know the sequence of spin systems along the molecular backbone. For example, spin system assignment is required to unambiguously assign NOE correlations for structural determination of folded forms in solution. Therefore, we developed a method to assign the aromatic spin systems in these compounds using HMBC experiments. This has been performed for tetrameric (Es4), pentameric (Es5), and hexameric (Es6) oligomers and is expected to prove useful for this class of foldamers in general. The proton assignments obtained by this technique have been useful toward confirming the previous hypotheses of helical folding in oPE systems.     

Total assignment and structure in solution of tetrandrine by NMR spectroscopy and molecular modelling

High-resolution 1- and 2D NMR spectra of tetrandrine and molecular modelling were employed to characterise its structure in solution. Complete and unambiguous assignment of all proton and carbon resonance signals is reported. Scalar couplings were determined from dihedral angles with the Karplus equation. Inter-proton distances were evaluated from NOE correlation peaks. Comparison of simulated and X-ray conformations of tetrandrine reveals only small differences.     

NMR elucidation of some ligands derived from the pentacycloundecane skeleton

The complete NMR elucidation of three novel pentacycloundecane (PCU)-derived ligands is reported. 2D NMR techniques are used to overcome the problem of major overlapping of methine signals on the cage skeleton. The compounds were synthesized as potential ligands to be used in asymmetric catalysis. They represent the first instance where aromatic moieties have been attached directly to the cage skeleton using lithiation techniques. The X-ray crystal structure of one of the ligands was obtained. The X-ray structure was helpful in determining the potential NOESY interactions within the set of molecules. For the other ligands a high level Density Functional Theory (DFT) optimization was performed [B3LYP/6-31+g(d)] to visualize possible NOE interactions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stabilising Peptoid Helices Using Non‐Chiral Fluoroalkyl Monomers

Stability towards protease degradation combined with modular synthesis has made peptoids of considerable interest in the fields of chemical biology, medicine, and biomaterials. Given their tertiary amide backbone, peptoids lack the capacity to hydrogen‐bond, and as such, controlling secondary structure can be challenging. The incorporation of bulky, charged, or chiral aromatic monomers can be used to control conformation but such building blocks limit applications in many areas. Through NMR and X‐ray analysis we demonstrate that non‐chiral neutral fluoroalkyl monomers can be used to influence the K_cis/trans equilibria of peptoid amide bonds in model systems. The cis‐isomer preference displayed is highly unprecedented given that neither chirality nor charge is used to control the peptoid amide conformation. The application of our fluoroalkyl monomers in the design of a series of linear peptoid oligomers that exhibit stable helical structures is also reported.     

Phenanthroline dicarboxamide-based helical foldamers: stable helical structures in methanol

A series of new aromatic oligoamides 2-5 based on 1,10-phenanthroline diacid and o-phenylenediamine have been synthesized through a convergent segment coupling strategy. These oligomers can fold into well-defined helical structures in solution through intramolecular hydrogen bonds and aromatic stacking interactions, which has been established by 1H NMR, fluorescence, and UV/vis spectra. In particular, it was found that the oligomers were more favorable to fold into stable helical structures in methanol than in chloroform and dichloromethane. The helical foldamers formed in the solid state have been characterized by single-crystal X-ray diffraction analysis. The results showed that the high curvature of the strands led to one and a half turns for both 2 and 21, three turns for 4, and nearly four turns for 5.     

Structure Elucidator: a versatile expert system for molecular structure elucidation from 1D and 2D NMR data and molecular fragments

StrucEluc is an expert system that allows the computer-assisted elucidation of chemical structures based on the inputs of a series of spectral data including 1D and 2D NMR and mass spectra. The system has been enabled to allow a chemist to utilize fragments stored in a fragment database as well as user-defined fragments submitted by the chemist in the structure elucidation process. The association of fragments in this way has been shown to dramatically speed up the process of structure generation from 2D NMR data and has helped to minimize or eliminate the need for user intervention thereby further enabling the vision of automated elucidation. The use of fragments has frequently transformed very difficult 2D NMR elucidation challenges into easily solvable tasks. A strategy to utilize molecular fragments has been developed and optimized based on specific challenging examples. This strategy will be described here using real world examples. Experience gained by solving more than 150 structure elucidation problems from a variety of literature sources is also reviewed in this work.     

Automated structure elucidation — the benefits of a symbiotic relationship between the spectroscopist and the expert system

Characteristic features of a new expert system StrucEluc are described. The system is intended for the structure elucidation of complex organic molecules using a variety of spectroscopic data including 2D NMR. We review here the results of challenging this system with over 100 structure elucidation problems where the 2D NMR peak tables presented in original journal publications provided the input data. This contribution is focused on methods to overcome difficult situations that can arise when contradictions are present in the input data and/or when the structure is underdetermined as a result of insufficient 2D NMR correlations. Methods by which to address these situations are examined. It has been shown that synergy between the spectroscopist and the expert system allows the solution of problems that seemed to be hopeless at the outset of the structure elucidation process.     

Sequence-controlled oligomers fold into nanosolenoids and impart unusual optical properties

Controlled syntheses give unique block oligomers with alternating flexible ethylene glycol and rigid perylenetetracarboxylic diimide (PDI) units. The number of rigid units vary from n=1 to 10. PDI units were stitched together by using efficient phosphoramidite chemistry. The resulting oligomers undergo folding in most solvents, including chloroform. In their ground state, these folded oligomers were characterized by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS), NMR spectroscopy, and electronic absorption spectroscopy. FTICR‐MS revealed the exact masses of these sequence‐controlled oligomers, which confirmed the chemical composition and validated the synthetic strategy. The NMR neighboring ring‐current effect (NRE) indicates the formation of cofacial π stacks; the stacked aromatic rings have nearly coaxial alignment akin to a nanosoleniod. Nanosolenoidal shielding in π stacks causes all aromatic protons to shift upfield, whereas NOE in a cyclic hetero‐chromophoric dimer supports a rotated, cofacial π‐stacking orientation separated by about 3.5 Å. Electron–phonon coupling is much stronger than excitonic coupling in these self‐folded PDI oligomers; thus, Franck–Condon factors dictate the observed spectral features in visible spectra. The absorbance spectrum exhibits weak hypochromism due to π stacking with increasing stacking units n. Finally, ab initio calculations support the experimental observations, indicating 3.5 Å cofacial spacing in which one molecule is rotated 30° from the eclipsed orientation and higher oligomers can adopt, without a compensating energy penalty, either the right/left‐handed helices or the 1,3‐eclipsed structures. Both theory and experiments validate the nano‐π‐solenoids and their novel photophysical properties.     

The structure and conformational behavior of sulfonium salt glycosidase inhibitors in solution: a combined quantum mechanical NMR approach

We present the conformational analysis of an inhibitor of alpha-mannosidase, based on a novel sulfonium salt structure (1) that mimics the mannosyl cation intermediate. Because of the number of possible isomeric structures for 1, as well as its complex molecular structure, traditional conformational analysis by NMR was not applicable. Instead, a single experimentally consistent structure was obtained from finite perturbation quantum mechanical calculations of the NMR J-couplings at the B3LYP/6-311G** level. Using a full relaxation matrix analysis, we showed that the quantum-predicted NMR structure was the only isomer that was consistent with the experimental NOE intensities. The results illustrate the potential for finite perturbation calculations to be useful in the analysis of complex charged molecules.     

NMR studies of polymer solutions. II. Polyethylene

The intramolecular structure of polyethylene in solution was studied by a high-resolution nuclear magnetic resonance technique. Highly purified n-alkanes (99.5%) from C₅H₁₂ to C₃₆H₇₄ were used as its oligomers. The NMR spectra of the polyethylenes (oligomers) are very sensitive to the solvents used. The internal methylene protons of all polyethylenes of various chain length resonance at an identical frequency in carbon tetrachloride. A sharp transition in the NMR spectrum of polyethylene in α-chloronaphthalene at 35°C. was observed at n-C₁₇H₃₆, above which there exist two distinguishable NMR peaks for internal methylene protons, and below which (fewer carbons) only a single peak was seen. The NMR spectra of the internal methylene protons of the polyethylenes (oligomers) taken in benzene are very similar to those taken in pyridine. They are not as easily resolved as those NMR spectra taken in α-chloronaphthalene solutions. The effect of the size of the aromatic solvent molecule on the NMR spectra of the internal methylene protons of the polyethylenes (oligomers) in solutions was demonstrated by using aromatic solvents of various sizes, such as chlorobenzene, α-chloronaphthalene, and 9-chloronathracene. The results indicate that the formation of polymeric structure of the internal methylene groups in the polyethylene chain is very sensitive to the size of the solvent used. The interaction of the solvent with the methylene groups of the polyethylenes varies as a function of chain length; it is stronger for those low member n-alkanes and decreases gradually to an asymptotic value.     

Conformation and absolute configuration of nocathiacin I determined by NMR spectroscopy and chiral capillary electrophoresis

Nocathiacin I (BMS-249524) is a highly cross-linked thiazolyl peptide that displays potent activity against Gram-positive bacteria, including a number of antibiotic-resistant strains. This natural product contains 10 chiral centers. NMR studies have been performed to characterize the solution structure of nocathiacin I. A uniformly 13C,15N-labeled sample was used to obtain NMR assignments. Restrained simulated annealing calculations were performed by using accurately determined NOE distance restraints. All of the chiral centers were allowed to float during the simulated annealing protocol. Two clusters of structures were obtained that satisfy the NOE restraints very well and that are reasonably consistent with vicinal J-coupling constants. Within each cluster, all 10 chiral centers are uniquely defined. The two clusters are effectively mirror images of each other: all chiral centers that have the R(S) configuration in one cluster have the S(R) configuration in the other. The single threonine residue in nocathiacin I was subsequently determined to be l-threonine by chiral capillary electrophoresis, allowing the absolute configurations of all 10 chiral centers to be defined.     

BINOL-based foldamers--access to oligomers with diverse structural architectures

In this article, we report on the synthesis and conformation of a new family of aromatic oligoamide foldamers based on binaphthol (BINOL) monomers. A series of oligomers with differing chirality of the individual BINOL building blocks and mixed sequences of alternate BINOL and pyridyl building blocks has been synthesized and structurally characterized. NMR and quantum chemical calculations on the basis of ab initio MO theory were performed to obtain insight into the conformational features of these oligomers. It is shown that the combination of these inherently chiral aromatic building blocks provides a novel access to a wide variety of conformationally ordered synthetic oligomers with diverse and dazzling structural architectures distinct from those classically observed.     

固体核磁碳结构参数的修正及其在煤结构分析中的应用

为消除~(13)C CP/M AS/TOSS NM R测试中碳核NOE效应,获得相对准确的碳结构参数,考察了不同模型化合物的碳核NOE效应强度。结果表明,不同模型化合物碳谱分峰拟合的测试值与样品碳结构参数的理论值之间存在明显误差,其中,脂肪碳在25%-125%、芳香碳为4%-50%,NOE效应在固体核磁碳谱测试中影响显著。为此,将模型化合物脂肪碳和芳香碳的实测值和理论值进行回归分析,得到非线性回归方程。用该方程对9,10-二甲基蒽进行碳结构修正,发现修正后脂肪碳实测值与理论值之间误差由不修正时的119.60%减小至7.84%,芳香碳误差为由不修正时的-17.10%到1.11%,修正后误差均在10%以内;同时用该回归方程修正了不同煤的碳结构参数,发现不同煤未修正的H/C原子比与其元素分析H/C原子比误差在45%-53%,修正后误差只有4%-13%,与元素分析结果具有一致性,表明非线性回归方程能够方便、准确地消除固体核磁NOE效应,为煤中碳结构分析提供新的技术支撑。     

Designing Foldamer–Foldamer Interactions in Solution: The Roles of Helix Length and Terminus Functionality in Promoting the Self‐Association of Aminoisobutyric Acid Oligomers

The biological activity of antibiotic peptaibols has been linked to their ability to aggregate, but the structure–activity relationship for aggregation is not well understood. Herein, we report a systematic study of a class of synthetic helical oligomer (foldamer) composed of aminoisobutyric acid (Aib) residues, which mimic the folding behavior of peptaibols. NMR spectroscopic analysis was used to quantify the dimerization constants in solution, which showed hydrogen‐bond donors at the N terminus promoted aggregation more effectively than similar modifications at the C terminus. Elongation of the peptide chain also favored aggregation. The geometry of aggregation in solution was investigated by means of titrations with [D₆]DMSO and 2D NOE NMR spectroscopy, which allowed the NH protons most involved in intermolecular hydrogen bonds in solution to be identified. X‐ray crystallography studies of two oligomers allowed a comparison of the inter‐ and intramolecular hydrogen‐bonding interactions in the solid state and in solution and gave further insight into the geometry of foldamer–foldamer interactions. These solution‐based and solid‐state studies indicated that the preferred geometry for aggregation is through head‐to‐tail interactions between the N and C termini of adjacent Aib oligomers.