Chemical Shift Data Research Articles

Using NMR Chemical Shifts and Cryo-EM Density Restraints in Iterative Rosetta-MD Protein Structure Refinement.

Cryo-EM has become one of the prime methods for protein structure elucidation, frequently yielding density maps with near-atomic or medium resolution. If protein structures cannot be deduced unambiguously from the density maps, computational structure refinement tools are needed to generate protein structural models. We have previously developed an iterative Rosetta-MDFF protocol that used cryo-EM densities to refine protein structures. Here we show that, in addition to cryo-EM densities, incorporation of other experimental restraints into the Rosetta-MDFF protocol further improved refined structures. We used NMR chemical shift (CS) data integrated with cryo-EM densities in our hybrid protocol in both the Rosetta step and the molecular dynamics (MD) simulations step. In 15 out of 18 cases for all MD rounds, the refinement results obtained when density maps and NMR chemical shift data were used in combination outperformed those of density map-only refinement. Notably, the improvement in refinement was highest when medium and low-resolution density maps were used. With our hybrid method, the RMSDs of final models obtained were always better than the RMSDs obtained by our previous protocol with just density refinement for both medium (6.9 Å) and low (9 Å) resolution maps. For all the six test proteins with medium resolution density maps (6.9 Å), the final refined structure RMSDs were lower for the hybrid method than for the cryo-EM only refinement. The final refined RMSDs were less than 1.5 Å when our hybrid protocol was used with 4 Å density maps. For four out of the six proteins the final RMSDs were even less than 1 Å. This study demonstrates that by using a combination of cryo-EM and NMR restraints, it is possible to refine structures to atomic resolution, outperforming single restraint refinement. This hybrid protocol will be a valuable tool when only low-resolution cryo-EM density data and NMR chemical shift data are available to refine structures.

Conditional Prediction of Ribonucleic Acid Secondary Structure Using Chemical Shifts.

Inspired by methods that utilize chemical-mapping data to guide secondary structure prediction, we sought to develop a framework for using assigned chemical shift data to guide ribonucleic acid (RNA) secondary structure prediction. We first used machine learning to develop classifiers that predict the base-pairing status of individual residues in an RNA based on their assigned chemical shifts. Then, we used these base-pairing status predictions as restraints to guide RNA folding algorithms. Our results showed that we could recover the correct secondary fold of most of the 108 RNAs in our data set with remarkable accuracy. Finally, we tested whether we could use the base-pairing status predictions that we obtained from assigned chemical shift data to conditionally predict the secondary structure of RNA. To achieve this, we attempted to model two distinct conformational states of the microRNA-20b and the fluoride riboswitch using assigned chemical shifts that were available for both conformational states of each of these test RNAs. For both test cases, we found that by using the base-pairing status predictions that we obtained from assigned chemical shift data as folding restraints, we could generate structures that closely resembled the known structure of the two distinct states. A command-line tool for chemical shifts to base-pairing status predictions in RNA has been incorporated into our CS2Structure Git repository and can be accessed via https://github.com/atfrank/CS2Structure .

Molecular Dynamics model of peptide-protein conjugation: case study of covalent complex between Sos1 peptide and N-terminal SH3 domain from Grb2

We have investigated covalent conjugation of VPPPVPPRRRX′ peptide (where X′ denotes Nε-chloroacetyl lysine) to N-terminal SH3 domain from adapter protein Grb2. Our experimental results confirmed that the peptide first binds to the SH3 domain noncovalently before establishing a covalent linkage through reaction of X′ with the target cysteine residue C32. We have also confirmed that this reaction involves a thiolate-anion form of C32 and follows the SN2 mechanism. For this system, we have developed a new MD-based protocol to model the formation of covalent conjugate. The simulation starts with the known coordinates of the noncovalent complex. When two reactive groups come into contact during the course of the simulation, the reaction is initiated. The reaction is modeled via gradual interpolation between the two sets of force field parameters that are representative of the noncovalent and covalent complexes. The simulation proceeds smoothly, with no appreciable perturbations to temperature, pressure or volume, and results in a high-quality MD model of the covalent complex. The validity of this model is confirmed using the experimental chemical shift data. The new MD-based approach offers a valuable tool to explore the mechanics of protein-peptide conjugation and build accurate models of covalent complexes.

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis.

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature‐dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics. Fundamentally, these temperature dependences result from changes in the local magnetic environments of nuclei, but correlations with global thermodynamic parameters measured via calorimetric methods have been observed. Although the temperature dependences of amide proton and nitrogen chemical shifts are often well approximated by a linear model, deviations from linearity are also observed and may be interpreted as evidence of fast exchange between distinct conformational states. Here, we describe computational methods, accessible via the Shift‐T web server, including an automated tracking algorithm that propagates initial (single temperature) 1H—15N cross peak assignments to spectra collected over a range of temperatures. Amide proton and nitrogen temperature coefficients (slopes determined by fitting chemical shift vs. temperature data to a linear model) are subsequently calculated. Also included are methods for the detection of systematic, statistically significant deviation from linearity (curvature) in the temperature dependences of amide proton chemical shifts. The use and utility of these methods are illustrated by example, and the Shift‐T web server is freely available at http://meieringlab.uwaterloo.ca/shiftt.

Backbone and side-chain chemical shift assignments for the ribosome-inactivating protein trichobakin (TBK).

Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC50: 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC50: 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of 1H, 13C, and 15N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.

NMR resonance assignments of the four isoforms of the hazelnut allergen Cor a 1.04

In large parts of Europe, Northern America and China people are suffering from allergies after consuming certain kinds of fruits and vegetables. Typical allergic symptoms range from scratching and itching of the throat to severe symptoms like rhino conjunctivitis and anaphylaxis. For hazelnuts (Corylus avellana), these allergies result from initial sensitization to the birch (Betula verrucosa) pollen allergen Bet v 1 and subsequent development of allergic cross-reactions to proteins that are similar in their three-dimensional structure to the sensitizing protein Bet v 1. The cross-reactive proteins in hazelnut are the four isoforms Cor a 1.04 with a molecular weight of about 17.5 kDa. Significant differences regarding the immunologic behavior of these proteins have been reported. In this work we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of these four isoforms, Cor a 1.0401, Cor a 1.0402, Cor a 1.0403, and Cor a 1.0404 by solution NMR spectroscopy. The chemical shift data confirm the characteristic Bet v onefold for all four isoforms, consisting of seven β-strands that are separated by two short α-helices, along with a long C-terminal α-helix. These data provide the basis for a comparative structural and dynamic analysis of these proteins by NMR in order to characterize their different immunologic cross-reactivities on a molecular level.

CORRELATION BETWEEN PHARMACOKINETIC PROPERTIES AND 15N-NMR AND 13C-NMR CHEMICAL SHIFTS OF ANGIOTENSIN CONVERTING ENZYME INHIBITORS

In the current study, angiotensin converting enzyme inhibitor molecules (enalapril, perindopril, ramipril and benazepril) were studied using 15N-nuclear magnetic resonance techniques like insensitive nuclei enhanced by polarization technique and heteronuclear multiple bond correlation. the chemical shift data and different pharmacokinetic and physicochemical properties of the molecules like pKa, half-life, time taken to reach maximum concentration in vivo, were compared and found to exhibit a good linear relationship. Further, a similar comparative study of the same parameters was performed using 13c chemical shifts of the molecules. Molecular docking studies were also performed to understand the structure of the enzyme bound to the ligand. this is perhaps the first report of 15N-nuclear magnetic resonance studies of the molecules as well as studies correlating the relationships between 15N chemical shifts, pharmacokinetics and physicochemical properties in molecules which could be used for the prediction of properties for which experimental data is currently not available.

Classification of RNA backbone conformations into rotamers using 13C' chemical shifts: exploring how far we can go.

The conformational space of the ribose-phosphate backbone is very complex as it is defined in terms of six torsional angles. To help delimit the RNA backbone conformational preferences, 46 rotamers have been defined in terms of these torsional angles. In the present work, we use the ribose experimental and theoretical 13C′ chemical shifts data and machine learning methods to classify RNA backbone conformations into rotamers and families of rotamers. We show to what extent the experimental 13C′ chemical shifts can be used to identify rotamers and discuss some problem with the theoretical computations of 13C′ chemical shifts.

Bayesian-Maximum-Entropy Reweighting of IDP Ensembles Based on NMR Chemical Shifts

Bayesian and Maximum Entropy approaches allow for a statistically sound and systematic fitting of experimental and computational data. Unfortunately, assessing the relative confidence in these two types of data remains difficult as several steps add unknown error. Here we propose the use of a validation-set method to determine the balance, and thus the amount of fitting. We apply the method to synthetic NMR chemical shift data of an intrinsically disordered protein. We show that the method gives consistent results even when other methods to assess the amount of fitting cannot be applied. Finally, we also describe how the errors in the chemical shift predictor can lead to an incorrect fitting and how using secondary chemical shifts could alleviate this problem.

Spectroscopic (FT-IR, FT-Raman, NMR and UV-Vis), ELF, LOL, NBO, and Fukui function investigations on (5-bromo-benzofuran-3-yl)-acetic acid hydrazide (5BBAH): Experimental and theoretical approach

Experimental and theoretical spectroscopic studies were performed for the (5-Bromo-benzofuran-3-yl)-acetic acid hydrazide (5BBAH) molecule. The research work was performed in two levels. In the first level, experimental FT-IR, FT-Raman, NMR (1H, 13C) chemical shift and UV-Vis spectral data were recorded. In the next level the theoretical computations have been conducted from DFT/B3LYP/6-311++G (d, p) basis level. Initially, the theoretical geometrical parameters were obtained and matched with related experimental parameters. The computed FT-IR and FT-Raman frequencies were collected from same basis level and matched with experimental data. The vibrational assignments were achieved on the PED of individual vibrational modes. The theoretical NMR chemical shifts were compared with recorded experimental data. Theoretical (TD-DFT) and experimental UV-Vis absorption wavelengths (λ) were compared with each other. In addition, FMO, NBO, MEP, ELF, LOL and Fukui function analysis were performed.

Guaianolides from Ormenis mixta: Structural Insights and Evaluation of Their Anti-inflammatory Profile.

In this paper, the isolation of five new guaianolides (1: - 5: ) and four (6: - 9: ) known sesquiterpenes from Ormenis mixta aerial parts is reported. The structural determination of the guaianolides was obtained by NMR spectroscopic data, as well as MS experiments. Their relative configurations were assigned by a combined quantum mechanical/NMR approach, comparing the experimental 13C/1H NMR chemical shift data and 1 J H-H homonuclear coupling constants with the related predicted values. The isolates were assayed for their anti-inflammatory potential evaluating nitric oxide release and cyclooxygenase-2 expression in J774A.1 macrophages treated with lipopolysaccharide from Escherichia coli. Our results indicated that, among the tested compounds, 1: - 3: , and 7: were able to inhibit nitric oxide release, while all were able to inhibit cyclooxygenase-2 expression with different potencies.

Quality and bias of protein disorder predictors

Disorder in proteins is vital for biological function, yet it is challenging to characterize. Therefore, methods for predicting protein disorder from sequence are fundamental. Currently, predictors are trained and evaluated using data from X-ray structures or from various biochemical or spectroscopic data. However, the prediction accuracy of disordered predictors is not calibrated, nor is it established whether predictors are intrinsically biased towards one of the extremes of the order-disorder axis. We therefore generated and validated a comprehensive experimental benchmarking set of site-specific and continuous disorder, using deposited NMR chemical shift data. This novel experimental data collection is fully appropriate and represents the full spectrum of disorder. We subsequently analyzed the performance of 26 widely-used disorder prediction methods and found that these vary noticeably. At the same time, a distinct bias for over-predicting order was identified for some algorithms. Our analysis has important implications for the validity and the interpretation of protein disorder, as utilized, for example, in assessing the content of disorder in proteomes.

Solution NMR Analysis of Ligand Environment in Quaternary Ammonium-Terminated Self-Assembled Monolayers on Gold Nanoparticles: The Effect of Surface Curvature and Ligand Structure.

We report a solution NMR-based analysis of (16-mercaptohexadecyl)trimethylammonium bromide (MTAB) self-assembled monolayers on colloidal gold nanospheres (AuNSs) with diameters from 1.2 to 25 nm and gold nanorods (AuNRs) with aspect ratios from 1.4 to 3.9. The chemical shift analysis of the proton signals from the solvent-exposed headgroups of bound ligands suggests that the headgroups are saturated on the ligand shell as the sizes of the nanoparticles increase beyond ∼10 nm. Quantitative NMR shows that the ligand density of MTAB-AuNSs is size-dependent. Ligand density ranges from ∼3 molecules per nm2 for 25 nm particles to up to 5-6 molecules per nm2 in ∼10 nm and smaller particles for in situ measurements of bound ligands; after I2/I- treatment to etch away the gold cores, ligand density ranges from ∼2 molecules per nm2 for 25 nm particles to up to 4-5 molecules per nm2 in ∼10 nm and smaller particles. T2 relaxation analysis shows greater hydrocarbon chain ordering and less headgroup motion as the diameter of the particles increases from 1.2 nm to ∼13 nm. Molecular dynamics simulations of 4, 6, and 8 nm (11-mercaptoundecyl)trimethylammonium bromide-capped AuNSs confirm greater hydrophobic chain packing order and saturation of charged headgroups within the same spherical ligand shell at larger nanoparticle sizes and higher ligand densities. Combining the NMR studies and MD simulations, we suggest that the headgroup packing limits the ligand density, rather than the sulfur packing on the nanoparticle surface, for ∼10 nm and larger particles. For MTAB-AuNRs, no chemical shift data nor ligand density data suggest that two populations of ligands that might correspond to side-ligands and end-ligands exist; yet T2 relaxation dynamics data suggest that headgroup mobility depends on aspect ratio and absolute nanoparticle dimensions.

Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated [Formula: see text] oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 μs, K diss ∼ 0.1 M) that goes on to form a tetramer ([Formula: see text] μs; K diss ∼ 22 μM), or exchanges with a "nonproductive" dimer that does not oligomerize further (τex ∼ 400 μs; K diss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D 2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

Toward Structure Determination of Disulfide-Rich Peptides Using Chemical Shift-Based Methods.

Disulfide-rich peptides are a class of molecules for which NMR spectroscopy has been the primary tool for structural characterization. Here, we explore whether the process can be achieved by using structural information encoded in chemical shifts. We examine (i) a representative set of five cyclic disulfide-rich peptides that have high-resolution NMR and X-ray structures and (ii) a larger set of 100 disulfide-rich peptides from the PDB. Accuracy of the calculated structures was dependent on the methods used for searching through conformational space and for identifying native conformations. Although Hα chemical shifts could be predicted reasonably well using SHIFTX, agreement between predicted and experimental chemical shifts was sufficient for identifying native conformations for only some peptides in the representative set. Combining chemical shift data with the secondary structure information and potential energy calculations improved the ability to identify native conformations. Additional use of sparse distance restraints or homology information to restrict the search space also improved the resolution of the calculated structures. This study demonstrates that abbreviated methods have potential for elucidation of peptide structures to high resolution and further optimization of these methods, e.g., improvement in chemical shift prediction accuracy, will likely help transition these methods into the mainstream of disulfide-rich peptide structural biology.

Experimental and Computational Characterization of the Conformational Ensemble and Interaction Motifs of Chiz N-Terminal Intrinsically Disordered Region

Cell division of Mycobacterium tuberculosis is crucial for the pathogenesis of tuberculosis. This process is mediated by the divisome, a multi-component, dynamic complex anchored by transmembrane proteins. One such protein is ChiZ, which contains a single transmembrane helix preceded by a 64-residue N-terminal intrinsically disordered region (IDR). We have characterized the conformational ensemble and membrane interactions of the ChiZ IDR by a variety of experimental techniques, including small-angle X-ray scattering (SAXS) and solution and solid-state NMR. 2D 1H-13C INEPT and PRE experiments suggest that the N-terminal IDR of ChiZ remains dynamic in the presence of liposomes, and interactions with the lipid membrane are mediated by arginine residues. To help interpret these data with atomistic details, we have carried out multiple molecular dynamics (MD) simulations in water using several force fields tailored for disordered proteins. The best agreement with SAXS, chemical shift, and NMR relaxation data was obtained by AMBER14SB/Tip4pD, followed closely by AMBER03ws/Tip4p2005s. In the MD simulations, the IDR transiently populated helices, especially in the N-terminal half, including residues 11-15, 19-22, and 28-32. This result explains the lower amide proton exchange rates in the N-terminal half of the IDR observed by solution NMR. Simulations in the presence of a lipid bilayer, in line with the solid-state NMR data, showed multiple arginine residues interacting with the membrane, where any one arginine may engage 2 or 3 lipid headgroups at once. Ongoing work aims to determine the dominant conformations of the full-length ChiZ in the membrane environment.

GPU Accelerated Computation of Isotropic Chemical Shifts Offers New Dimension of Structure Refinement in Largescale Molecular Dynamics Simulation

Semi-empirical chemical shift prediction, where molecular coordinates and empirical data are reduced to backbone and sidechain chemical shifts through input to differentiable functions, offers a unique opportunity to validate and refine protein structure during largescale molecular dynamics (MD) simulation but poses the challenge of optimizing performance to levels comparable to modern GPU-enabled MD engines. The software PPM_One predicts backbone and sidechain chemical shifts with competitive accuracy to purely data-driven counterparts while obviating the need for deep network processing and homology assignment. To poise PPM_One for practical application in largescale biomolecular modeling, a newly optimized GPU-accelerated version of PPM_One was developed. Code refactoring and parallel GPU-processing through the directive-based OpenACC API provide a 67-fold speedup between unaccelerated and Nvidia V100 accelerated PPM_One test-cases, comparatively, in a system of 21 million protein atoms. The core functions that comprise the prediction model and compute perturbation due to Hydrogen bonding, magnetic anisotropy and ring currents were profiled at 271x, 322x, and 211x speedups, respectively. Remaining components of the predictive model saw similar individual speedups through profiling. Implementing the newly optimized functional core into NAMD is NMRForces, a global force module that enables users to compute backbone and sidechain chemical shifts at each timestep and force atoms along the prediction model's gradient with a magnitude proportional to deviance from experimental chemical shift data.

Solid-State NMR and MD Study of the Structure of the Statherin Mutant SNa15 on Mineral Surfaces.

Elucidation of the structure and interactions of proteins at native mineral interfaces is key to understanding how biological systems regulate the formation of hard tissue structures. In addition, understanding how these same proteins interact with non-native mineral surfaces has important implications for the design of medical and dental implants, chromatographic supports, diagnostic tools, and a host of other applications. Here, we combine solid-state NMR spectroscopy, isotherm measurements, and molecular dynamics simulations to study how SNa15, a peptide derived from the hydroxyapatite (HAP) recognition domain of the biomineralization protein statherin, interacts with HAP, silica (SiO2), and titania (TiO2) mineral surfaces. Adsorption isotherms are used to characterize the binding affinity of SNa15 to HAP, SiO2, and TiO2. We also apply 1D 13C CP MAS, 1D 15N CP MAS, and 2D 13C-13C DARR experiments to SNa15 samples with uniformly 13C- and 15N-enriched residues to determine backbone and side-chain chemical shifts. Different computational tools, namely TALOS-N and molecular dynamics simulations, are used to deduce secondary structure from backbone and side-chain chemical shift data. Our results show that SNa15 adopts an α-helical conformation when adsorbed to HAP and TiO2, but the helix largely unravels upon adsorption to SiO2. Interactions with HAP are mediated in general by acidic and some basic amino acids, although the specific amino acids involved in direct surface interaction vary with surface. The integrated experimental and computational approach used in this study is able to provide high-resolution insights into adsorption of proteins on interfaces.

Conformational Properties Relevant to the Amyloidogenicity of β2-Microglobulin Analyzed Using Pressure- and Salt-Dependent Chemical Shift Data

β2-Microglobulin (β2m) is associated with dialysis-related amyloidosis. In vitro experiments have shown that β2m forms amyloid fibrils at acidic pHs in the presence of moderate concentrations of salt. Previous studies suggested that acid-denatured β2m has a hydrophobic residual structure, and the exposure of the hydrophobic residues enhances the association with seeds or other β2m monomers. However, the nature of the residual structure relevant to its amyloidogenicity remains to be investigated. To understand the structural properties of acid-denatured β2m and the role of salt, we investigated pressure- and salt concentration-dependent conformational changes by nuclear magnetic resonance spectroscopy and other methods. Here, pressure was utilized to characterize the conformers existing in a conformational equilibrium at ambient pressure. The obtained pressure- and salt concentration-dependent chemical shift data were simultaneously subjected to principal component analysis to characterize individual conformational change events. Unexpectedly, the addition of salt induced an expansion of the β2m molecule, which likely resulted from the exclusion of the N-terminal region from the hydrophobic cluster region. The dissected chemical shift patterns for the salt-induced conformational change and other experimental data indicated that this conformational change caused a rigidification in the intrinsic hydrophobic cluster, leading to the observed amyloidogenicity.

Two non-identical twins in one unit cell: characterization of 34π aromatic core-modified octaphyrins, their structural isomers and anion bound complexes.

Four different core-modified planar 34π octaphyrins (10, 11, 13, and 15) which exhibit rotational isomerism have been synthesized and characterized both in solution and solid states. Octaphyrins 10, 11 and 13 show two inseparable isomers A and B which crystallize in the same unit cell. However, 15 forms two identical isomers of A. Structurally, the two isomers in 10, 11 and 13 (A and B) are different only in the ring inversion of one of the thiophene or selenophene rings present in the terthiophene subunit of the macrocycle. In isomer A, the middle thiophene or selenophene rings are inverted, while in isomer B, the terminal thiophene rings are inverted. The 1H NMR spectrum of these macrocycles shows peaks assignable to protons of both the isomers in toluene D8. The single crystal structure analysis of 10 reveals the presence of both isomers 10A and 10B in a single unit cell with the P21/n space group. Both the isomers exhibit aromatic behaviour in the freebase form. Protonation of pyrrole nitrogens leads to exclusive formation of isomer B for 10 and 11. However, both the isomers are present upon protonation of 13 where the central heterocyclic ring of terthiophene subunits has thiophene and selenophene rings. Octaphyrin 15 crystallizes in the P21/c space group and exclusively isomer A was formed in the reaction. Protonation of pyrrole nitrogens leads to significant increases in aromaticity as revealed by 1H NMR chemical shift data. The NICS values calculated for the individual heterocyclic rings before and after protonation support such a conclusion. The AICD plots exhibit clockwise orientation of current density vectors suggesting the presence of diatropic ring current in the octaphyrins. Energy calculations at the M06L/CC-pVTZ//M06L/6-31G** level qualitatively account for exclusive stabilization of a particular isomer relative to the other upon protonation. To the best of our knowledge 10 represents the first example in expanded porphyrin chemistry where two different structural isomers crystallize in a single unit cell.