Innovations in generation and analysis of 2D [(13)C,(1)H] COSY NMR spectra for metabolic flux analysis purposes.

2D [(13)C,(1)H] COSY NMR is used by the metabolic engineering community for determining (13)C-(13)C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [(13)C,(1)H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range (13)C-(13)C scalar couplings in 2D [(13)C,(1)H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed 13C NMR

13C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane alphaII-helices. Surprisingly, the 13C NMR spectra of [3-(13)C]Ala-D85N turned out to be very similar to those of [3-(13)C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting 13C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane alphaII-helices of the M-like state are suppressed already by fluctuation motions in the order of 10(4)-10(5) Hz interfered with frequencies of magic angle spinning or proton decoupling. However, 13C NMR signal from the cytoplasmic alpha-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl 13C NMR signals of the loop and transmembrane alpha-helices followed by Pro residues in [1-(13)C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, 13C NMR spectral feature of reconstituted [1-(13)C]Val and [3-(13)C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

A new polyamine, thermospermine, 1,12-diamino-4,8-diazadodecane, from an extreme thermophile.

A new polyamine has been extracted from an extreme thermophile, Thermus thermophilus, and its chemical structure was determined as 1,12-diamino-4,8-diazadodecane, NH2(CH2)3NH(CH2)3NH(CH2)4NH2, based on its proton NMR, 13C NMR, and mass spectra. A trivial name "thermospermine" is proposed for the new compound.     

Facile detection of protein-protein interactions by one-dimensional NMR spectroscopy

Two methods for detecting protein-protein interactions in solution using one-dimensional (1D) NMR spectroscopy are described. Both methods rely on measurement of the intensity of the strongest methyl resonance (SMR), which for most proteins is observed at 0.8-0.9 ppm. The severe resonance overlap in this region facilitates detection of the SMR at low micromolar and even sub-micromolar protein concentrations. A decreased SMR intensity in the 1H NMR spectrum of a protein mixture compared to the added SMR intensities of the isolated proteins reports that the proteins interact (SMR method). Decreased SMR intensities in 1D 13C-edited 1H NMR spectra of 13C-labeled proteins upon addition of unlabeled proteins or macromolecules also demonstrate binding (SMRC method). Analysis of the interaction between XIAP and Smac, two proteins involved in apoptosis, illustrates both methods. A study showing that phospholipids compete with the neuronal core complex for Ca2+-dependent binding to the presynaptic Ca2+-sensor synaptotagmin 1 illustrates the usefulness of the SMRC method in studying multicomponent systems.     

Multi-component analysis of marine lipids in fish gonads with emphasis on phospholipids using high resolution NMR spectroscopy

High resolution NMR has been applied for assessment of lipid classes and acyl stereospecific positions of fatty acids in marine phospholipids and triacylglycerols. 1D and 2D NMR techniques in combination with recording of a number of reference standards have been used to interpret the (1)H and (13)C NMR spectra of fish gonads. (13)C NMR spectra gave information regarding the polyunsaturated fatty acids (PUFAs) in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The carbonyl resonances showed that n-3 PUFAs primarily were esterified in the sn-2 position of PC and PE. The glycerol resonances showed that the PC/PE ratio was higher in roe than in milt and that roe comprised more triacylglycerols than milt. Thin layer chromatography showed that milt contained 2.4 times more cholesterol than roe, which was also found by integrating the (1)H NMR spectra. Concentration (mol%) of n-3 fatty acids were calculated from the (1)H NMR data and showed 44.8 and 36.3% in roe and milt, respectively.     

NMR analysis and sequence of toxin II from the sea anemone Radianthus paumotensis

Toxin II from Radianthus paumotensis (RpII) has been investigated by high-resolution NMR and chemical sequencing methods. Resonance assignments have been obtained for this protein by the sequential approach. NMR assignments could not be made consistent with the previously reported primary sequence for this protein, and chemical methods have been used to determine a sequence with which the NMR data are consistent. Analysis of the 2D NOE spectra shows that the protein secondary structure is comprised of two sequences of beta-sheet, probably joined into a distorted continuous sheet, connected by turns and extended loops, without any regular alpha-helical segments. The residues previously implicated in activity in this class of proteins, D8 and R13, occur in a loop region.     

2D NMR metabonomic analysis: a novel method for automated peak alignment

MOTIVATION: Comparative metabolic profiling by nuclear magnetic resonance (NMR) is showing increasing promise for identifying inter-individual differences to drug response. Two dimensional (2D) (1)H (13)C NMR can reduce spectral overlap, a common problem of 1D (1)H NMR. However, the peak alignment tools for 1D NMR spectra are not well suited for 2D NMR. An automated and statistically robust method for aligning 2D NMR peaks is required to enable comparative metabonomic analysis using 2D NMR. RESULTS: A novel statistical method was developed to align NMR peaks that represent the same chemical groups across multiple 2D NMR spectra. The degree of local pattern match among peaks in different spectra is assessed using a similarity measure, and a heuristic algorithm maximizes the similarity measure for peaks across the whole spectrum. This peak alignment method was used to align peaks in 2D NMR spectra of endogenous metabolites in liver extracts obtained from four inbred mouse strains in the study of acetaminophen-induced liver toxicity. This automated alignment method was validated by manual examination of the top 50 peaks as ranked by signal intensity. Manual inspection of 1872 peaks in 39 different spectra demonstrated that the automated algorithm correctly aligned 1810 (96.7%) peaks. AVAILABILITY: Algorithm is available upon request.     

Synthesis and anticancer activity of novel chiral D-glucose derived bis-imidazoles and their analogs.

A series of novel D-glucose derived bis-imidazoles and their analogs, which possess potential in bioorganic and supramolecular chemistry, were designed and synthesized from methyl alpha-D-glucoside through protection, bis-bromination, N-alkylation and deprotection. All new compounds were characterized by HRMS, 1H, 13C and DEPT NMR spectroscopy as well as elemental analysis. The 1H-(1)H and 1H-(13)C 2D NMR spectra for some compounds were also recorded. Some regular features of 13C and 1H NMR spectra were summarized. The anticancer activity of some compounds was evaluated.     

Automated protein fold determination using a minimal NMR constraint strategy

Determination of precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium-accuracy fold information can often provide key clues about protein evolution and biochemical function(s). In this article we describe a largely automatic strategy for rapid determination of medium-accuracy protein backbone structures. Our strategy derives from ideas originally introduced by other groups for determining medium-accuracy NMR structures of large proteins using deuterated, (13)C-, (15)N-enriched protein samples with selective protonation of side-chain methyl groups ((13)CH(3)). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side-chain (15)N, H(N) resonances, and side-chain (13)CH(3) methyl resonances. These assignments are determined automatically by the program AutoAssign using backbone triple resonance NMR data, together with Spin System Type Assignment Constraints (STACs) derived from side-chain triple-resonance experiments. The program AutoStructure then derives conformational constraints using these chemical shifts, amide (1)H/(2)H exchange, nuclear Overhauser effect spectroscopy (NOESY), and residual dipolar coupling data. The total time required for collecting such NMR data can potentially be as short as a few days. Here we demonstrate an integrated set of NMR software which can process these NMR spectra, carry out resonance assignments, interpret NOESY data, and generate medium-accuracy structures within a few days. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A.     

Comparative structural connectivity spectra analysis (CoSCoSA) models of steroids binding to the aromatase enzyme

A method that combines NMR spectral and structural information into a constructed three-dimensional (3D)-connectivity matrix is developed for modeling biological binding activity of small molecules. The 3D-connectivity matrix for a molecule is defined by associating the distances between all possible carbon-to-carbon connections with their assigned carbon NMR chemical shifts. In this project we selected from the total 3D-connectivity matrix a subset, the two-dimensional (2D) (13)C-(13)C COSY and a theoretical long range 2D (13)C-(13)C distance connectivity spectral plane. Patterns of (13)C chemical shifts observed at these two relative distances for 50 steroids were used to produce a mathematical relationship for the steroids' relative binding affinity (pK(i)) to the aromatase enzyme. We call this technique comparative structural connectivity spectra analysis (CoSCoSA) modeling. Using combinations of the 2D COSY and 2D long-range distance spectra as modeling parameters, we built four CoSCoSA models. One model was made from the 2D COSY spectra alone and another was developed using only the 2D long-range distance spectra. Then the COSY and long-distance spectra were combined in two different ways: starting with the combined principal components (PCs) from the separately calculated COSY and distance spectra or using the combined raw spectra (3D). The best CoSCoSA model was based on the combined PCs from COSY and distance spectra. This model had an r(2) of 0.96 and a leave-one-out cross-validation (q(2)) of 0.92. In general CoSCoSA modeling combines the quantum mechanical information inherent in NMR chemical shifts with internal molecular atom-to-atom distances to give a reliable and straightforward basis for predictive modeling. The technique has the flexibility and accuracy to outperform not only the cross-validated variance q(2) of previously published quantitative structure-activity relationships (QSAR) but also those obtained by related quantitative spectral data-activity relationships (QSDARs) lacking connectivity dimensions.     

Advances towards resonance assignments for uniformly—13C, 15N enriched bacteriorhodopsin at 18.8 T in purple membranes

Solid state NMR spectra from uniformly (13)C, (15)N enriched bacteriorhodospin (bR) purified from H. salinarium were acquired at 18.8 T using magic angle spinning methods. Isolated resonances of 2D (13)C-(13)C spectra exhibited 0.50-0.55 ppm line-widths. Several amino acid types could be assigned, and at least 12 out of 15 Ile peaks could be resolved clearly and identified based on their characteristic chemical shifts and connectivities. This study confirms that high resolution solid state NMR spectra can be obtained for a 248 amino acid uniformly labeled membrane protein in its native membrane environment and indicates that site-specific assignments are likely to be feasible with heteronuclear multidimensional spectra.     

1H NMR studies on bovine cyclophilin: preliminary structural characterization of its complex with cyclosporin A

Cyclophilin (163 residues, Mr 17737), a peptidyl prolyl cis-trans isomerase, is a cytosolic protein that specifically binds the potent immunosuppressant cyclosporin A (CsA). The native form of the major bovine thymus isoform has been analyzed by 2D NMR methods, COSY, HOHAHA, and NOESY, in aqueous media. The 156 main-chain amides in CyP yield 126 observable NH/alpha CH couplings (81%, Gly pairs counted as 1). Following exhaustive D2O exchange, 44 amide resonances remain visible. Further analysis of the NH/NH, NH/alpha CH, and alpha CH/alpha CH regions of the COSY and NOESY data sets indicates that the residual amides in D2O form a coherent hydrophobic domain which yields 2D NMR features suggestive of a beta-sheet. Many (43/126) of the amide resonances have been classified according to amino acid type. In the aromatic region of the spectra, the assignment of the ring spin systems is nearly complete (12/15 Phe, 2/2 Tyr, 1/1 Trp, and 3/4 His). This has successfully lead to the complete assignment of all of their beta CH's, main-chain alpha CH resonances, and many of the backbone amide resonances (8/12 Phe, 2/2 Tyr, 1/1 Trp, and 2/3 His). In other regions of the spectrum, the side-chain and main-chain resonances for 10/23 Gly, 9/9 Ala, 5/11 Thr, 5/9 Val, and 1/6 Leu have been completely assigned. The drug-free cyclophilin and CsA-bound cyclophilin form two discrete protein structures that are in slow exchange on the NMR time scale. Comparison of the fingerprint regions from the COSY spectra obtained from the two forms of the protein reveals a minimum of 16 cross-peaks which are clearly shifted upon complexation. In fact, on the basis of chemical shift changes observed in assigned side-chain and main-chain resonances, only a relatively few of the amino acid residues identified to date are perturbed by complex formation. These include 3 Phe (8, 12, and 14) and the Trp in the aromatic region and 2 Ala (7 and 8) in the Ala/Thr region. In the upfield-shifted methyl region, an assigned Leu and Val spin system and a spin system labeled X10 (an Ile or Leu) are affected by complex formation. In addition, a new aliphatic spin system, labeled X11, which shows a close spatial relationship to the perturbed Phe12, is observed in this region of the spectrum. In summary, the regions of the protein altered by complex formation can be divided into two categories: a hydrophobic and a H2O-accessible domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Innovations in Generation and Analysis of 2D [C,H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

2D [¹³C,¹H] COSY NMR is used by the metabolic engineering community for determining ¹³C–¹³C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [¹³C,¹H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range ¹³C–¹³C scalar couplings in 2D [¹³C,¹H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Lactoperoxidase-catalyzed oxidation of thiocyanate by hydrogen peroxide: a reinvestigation of hypothiocyanite by nuclear magnetic resonance and optical spectroscopy

In an effort to reconcile conflicting reports regarding the spectra of the human defense factor hypothiocyanite (OSCN(-)), we have synthesized OSCN(-) by three methods and characterized the product spectroscopically. Method I is lactoperoxidase-catalyzed oxidation of SCN(-) by H(2)O(2) at pH 7. Method II is hydrolysis of (SCN)(2) at pH 13. Method III is oxidation of SCN(-) by OX(-) (X = Cl and Br) at pH 13. All three methods produced essentially the same initial UV, (13)C NMR, and (15)N NMR spectra. The UV spectrum reveals a lambda(max) of 376 nm, which is a previously unreported distinguishing feature. The (13)C NMR spectrum (delta = 127.8 ppm at pH 13 vs dioxane at 66.6 ppm) is comparable to those that have been previously reported for OSCN(-) as prepared by methods I and II (although in some cases different assignments have been made). However, the (15)N NMR spectrum we measure (delta = -80.6 ppm at pH 13 vs NO(3)(-) at 0 ppm) contrasts with previous reports. We conclude that all three methods produce the same species, and the spectra are now self-consistent with the formulation OSCN(-).     

1H NMR assignment and melting temperature study of cis-syn and trans-syn thymine dimer containing duplexes of d(CGTATTATGC).d(GCATAATACG)

The preparation and spectroscopic characterization of duplex decamers containing site-specific cis-syn and trans-syn thymine dimers are described. Three duplex decamers, d(CGTATTATGC).d(GCATAATACG), d(CGTAT[c,s]TATGC).d(GCATAATACG), and d(CGTAT[t,s]TATGC).d(GCATAATACG), were prepared by solid-phase phosphoramidite synthesis utilizing cis-syn and trans-syn cyclobutane thymine dimer building blocks (Taylor et al., 1987; Taylor & Brockie, 1988). NMR spectra (500 MHz 2D 1H and 202 MHz 1D 31P) were obtained in "100%" D2O at 10 degrees C, and 1D exchangeable 1H spectra were obtained in 10% D2O at 10 degrees C. 1H NMR assignments for H5, H6, H8, CH3, H1', H2', and H2" were made on the basis of standard sequential NOE assignment strategies and verified in part by DQF COSY data. Comparison of the chemical shift data suggests that the helix structure is perturbed more to the 3'-side of the cis-syn dimer and more to the 5'-side of the trans-syn dimer. Thermodynamic parameters for the helix in equilibrium coil equilibrium were obtained by two-state, all or none, analysis of the melting behavior of the duplexes. Analysis of the temperature dependence of the T5CH3 1H NMR signal gave delta H = 44 +/- 4 kcal and delta S = 132 +/- 13 eu for the trans-syn duplex. Analysis of the concentration and temperature dependence of UV spectra gave delta H = 64 +/- 6 kcal and delta S = 178 +/- 18 eu for the parent duplex and delta H = 66 +/- 7 kcal and delta S = 189 +/- 19 eu for cis-syn duplex. It was concluded that photodimerization of the dTpdT unit to give the cis-syn product causes little perturbation of the DNA whereas dimerization to give the trans-syn product causes much greater perturbation, possibly in the form of a kink or dislocation at the 5'-side of the dimer.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy

The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13C and 15N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three-dimensional (3D) correlation experiments on a uniformly 13C, 15N-labeled sample at 750-MHz 1H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.     

C-13 NMR spectral studies of the thyroid hormone transport protein, transthyretin and the pancreatic insulin storage moiety, the zinc-insulin hexamer

The DEPT spectral editing technique was used to separate the CH, CH2 and CH3 resonances in the C-13 NMR spectra of transthyretin and the porcine zinc insulin hexamer. The advantages of this technique in terms of spectral simplification and sensitivity enhancement for 13C NMR of proteins is discussed. Spin-lattice relaxation time and nuclear Overhauser effect measurements of the backbone C-alpha and aliphatic sidechain carbons provided information about the dynamics of the proteins in solution and the relative mobility of some sidechain groups.     

A fast NMR method for resonance assignments: application to metabolomics

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (¹²C) or ¹³C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [¹³C, ¹H] HSQC–TOCSY, (2) 2D ¹H–¹H TOCSY and (3) 2D ¹³C–¹H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [¹³C, ¹H] HSQC–TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete ¹H and ¹³C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.     

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

The wild‐type HIV‐1 capsid protein (CA) self‐assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV‐1. Mass‐per‐length values of CA assemblies determined by dark‐field transmission electron microscopy indicate a variety of structures, ranging from single‐wall tubes to multiwall tubes that approximate solid rods. Two‐dimensional (2D) solid state ¹³C? ¹³C and ¹⁵N? ¹³C NMR spectra of uniformly ¹⁵N,¹³C‐labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3‐¹³C₂‐glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N‐terminal (NTD) and C‐terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self‐assembly of full‐length CA into tubes. 2D ¹H‐¹³C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the α‐helices of NTD and CTD and from the N‐ and C‐terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.