Backbone amide 15N chemical shift tensors report on hydrogen bonding interactions in proteins: A magic angle spinning NMR study.

Chemical shift tensors (CSTs) are an exquisite probe of local geometric and electronic structure. 15N CST are very sensitive to hydrogen bonding, yet they have been reported for very few proteins to date. Here we present experimental results and statistical analysis of backbone amide 15N CSTs for 100 residues of four proteins, two E. coli thioredoxin reassemblies (1-73-(U-13C,15N)/74-108-(U-15N) and 1-73-(U-15N)/74-108-(U-13C,15N)), dynein light chain 8 LC8, and CAP-Gly domain of the mammalian dynactin. The 15N CSTs were measured by a symmetry-based CSA recoupling method, ROCSA. Our results show that the principal component δ11 is very sensitive to the presence of hydrogen bonding interactions due to its unique orientation in the molecular frame. The downfield chemical shift change of backbone amide nitrogen nuclei with increasing hydrogen bond strength is manifested in the negative correlation of the principal components with hydrogen bond distance for both α-helical and ß-sheet secondary structure elements. Our findings highlight the potential for the use of 15N CSTs in protein structure refinement.

Sensitivity gains, linearity, and spectral reproducibility in nonuniformly sampled multidimensional MAS NMR spectra of high dynamic range.

Recently, we have demonstrated that considerable inherent sensitivity gains are attained in MAS NMR spectra acquired by nonuniform sampling (NUS) and introduced maximum entropy interpolation (MINT) processing that assures the linearity of transformation between the time and frequency domains. In this report, we examine the utility of the NUS/MINT approach in multidimensional datasets possessing high dynamic range, such as homonuclear (13)C-(13)C correlation spectra. We demonstrate on model compounds and on 1-73-(U-(13)C,(15)N)/74-108-(U-(15)N) E. coli thioredoxin reassembly, that with appropriately constructed 50% NUS schedules inherent sensitivity gains of 1.7-2.1-fold are readily reached in such datasets. We show that both linearity and line width are retained under these experimental conditions throughout the entire dynamic range of the signals. Furthermore, we demonstrate that the reproducibility of the peak intensities is excellent in the NUS/MINT approach when experiments are repeated multiple times and identical experimental and processing conditions are employed. Finally, we discuss the principles for design and implementation of random exponentially biased NUS sampling schedules for homonuclear (13)C-(13)C MAS correlation experiments that yield high-quality artifact-free datasets.

Multidimensional magic angle spinning NMR spectroscopy for site-resolved measurement of proton chemical shift anisotropy in biological solids.

The proton chemical shift (CS) tensor is a sensitive probe of structure and hydrogen bonding. Highly accurate quantum-chemical protocols exist for computation of (1)H magnetic shieldings in the various contexts, making proton chemical shifts potentially a powerful predictor of structural and electronic properties. However, (1)H CS tensors are not yet widely used in protein structure calculation due to scarcity of experimental data. While isotropic proton shifts can be readily measured in proteins even in the solid state, determination of the (1)H chemical shift anisotropy (CSA) tensors remains challenging, particularly in molecules containing multiple proton sites. We present a method for site-resolved measurement of amide proton CSAs in fully protonated solids under magic angle spinning. The approach consists of three concomitant 3D experiments yielding spectra determined by either mainly (1)H CSA, mainly (1)H­(15)N dipolar, or combined (1)H CSA and (1)H­(15)N dipolar interactions. The anisotropic interactions are recoupled using RN-sequences of appropriate symmetry, such as R12(1)(4), and (15)N/(13)C isotropic CS dimensions are introduced via a short selective (1)H­(15)N cross-polarization step. Accurate (1)H chemical shift tensor parameters are extracted by simultaneous fit of the lineshapes recorded in the three spectra. An application of this method is presented for an 89-residue protein, U-(13)C,(15)N-CAP-Gly domain of dynactin. The CSA parameters determined from the triple fits correlate with the hydrogen-bonding distances, and the trends are in excellent agreement with the prior solution NMR results. This approach is generally suited for recording proton CSA parameters in various biological and organic systems, including protein assemblies and nucleic acids.

Enhanced sensitivity by nonuniform sampling enables multidimensional MAS NMR spectroscopy of protein assemblies.

We report dramatic sensitivity enhancements in multidimensional MAS NMR spectra by the use of nonuniform sampling (NUS) and introduce maximum entropy interpolation (MINT) processing that assures the linearity between the time and frequency domains of the NUS acquired data sets. A systematic analysis of sensitivity and resolution in 2D and 3D NUS spectra reveals that with NUS, at least 1.5- to 2-fold sensitivity enhancement can be attained in each indirect dimension without compromising the spectral resolution. These enhancements are similar to or higher than those attained by the newest-generation commercial cryogenic probes. We explore the benefits of this NUS/MaxEnt approach in proteins and protein assemblies using 1-73-(U-(13)C,(15)N)/74-108-(U-(15)N) Escherichia coli thioredoxin reassembly. We demonstrate that in thioredoxin reassembly, NUS permits acquisition of high-quality 3D-NCACX spectra, which are inaccessible with conventional sampling due to prohibitively long experiment times. Of critical importance, issues that hinder NUS-based SNR enhancement in 3D-NMR of liquids are mitigated in the study of solid samples in which theoretical enhancements on the order of 3-4 fold are accessible by compounding the NUS-based SNR enhancement of each indirect dimension. NUS/MINT is anticipated to be widely applicable and advantageous for multidimensional heteronuclear MAS NMR spectroscopy of proteins, protein assemblies, and other biological systems.

Solid-state NMR spectroscopy of protein complexes.

Protein-protein interactions are vital for many biological processes. These interactions often result in the formation of protein assemblies that are large in size, insoluble, and difficult to crystallize, and therefore are challenging to study by structure biology techniques, such as single crystal X-ray diffraction and solution NMR spectroscopy. Solid-state NMR (SSNMR) spectroscopy is emerging as a promising technique for studies of such protein assemblies because it is not limited by molecular size, solubility, or lack of long-range order. In the past several years, we have applied magic angle spinning SSNMR-based methods to study several protein complexes. In this chapter, we discuss the general SSNMR methodologies employed for structural and dynamics analyses of protein complexes with specific examples from our work on thioredoxin reassemblies, HIV-1 capsid protein assemblies, and microtubule-associated protein assemblies. We present protocols for sample preparation and characterization, pulse sequences, SSNMR spectra collection, and data analysis.

Solid-state nuclear magnetic resonance studies delineate the role of the protein in activation of both aromatic rings of thiamin.

Knowledge of the state of ionization and tautomerization of heteroaromatic cofactors when enzyme-bound is essential for formulating a detailed stepwise mechanism via proton transfers, the most commonly observed contribution to enzyme catalysis. In the bifunctional coenzyme, thiamin diphosphate (ThDP), both aromatic rings participate in catalysis, the thiazolium ring as an electrophilic covalent catalyst and the 4'-aminopyrimidine as acid-base catalyst involving its 1',4'-iminopyrimidine tautomeric form. Two of four ionization and tautomeric states of ThDP are well characterized via circular dichroism spectral signatures on several ThDP superfamily members. Yet, the method is incapable of providing information about specific proton locations, which in principle may be accessible via NMR studies. To determine the precise ionization/tautomerization states of ThDP during various stages of the catalytic cycle, we report the first application of solid-state NMR spectroscopy to ThDP enzymes, whose large mass (160,000-250,000 Da) precludes solution NMR approaches. Three de novo synthesized analogues, [C2,C6'-(13)C(2)]ThDP, [C2-(13)C]ThDP, and [N4'-(15)N]ThDP used with three enzymes revealed that (a) binding to the enzymes activates both the 4'-aminopyrimidine (via pK(a) elevation) and the thiazolium rings (pK(a) suppression); (b) detection of a pre-decarboxylation intermediate analogue using [C2,C6'-(13)C(2)]ThDP, enables both confirmation of covalent bond formation and response in 4'-aminopyrimidine ring's tautomeric state to intermediate formation, supporting the mechanism we postulate; and (c) the chemical shift of bound [N4'-(15)N]ThDP provides plausible models for the participation of the 1',4'-iminopyrimidine tautomer in the mechanism. Unprecedented detail is achieved about proton positions on this bifunctional coenzyme on large enzymes in their active states.

A novel disulfide-rich protein motif from avian eggshell membranes.

Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4)-C-X(5)-C-X(8)-C-X(6) pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.

Solid-state NMR and density functional theory studies of ionization states of thiamin.

Thiamin diphosphate (ThDP) is a key coenzyme in sugar metabolism. The 4'-aminopyrimidine ring of ThDP cycles through several ionization and tautomeric states during enzyme catalysis, but it is not fully understood which states are adopted during the individual steps of the catalytic cycle. Thiamin has been synthesized with labels selectively inserted into the C2 and C6' positions, as well as into the amino group, creating [C2, C6'-(13)C(2)] thiamin and [N4'-(15)N] thiamin. Magic-angle spinning (MAS) NMR spectroscopy has been employed to record the (13)C and (15)N chemical shift anisotropy (CSA) tensors for C2, C6', and N4' atoms. Our results indicate that the isotropic chemical shifts as well as the principal components of the (13)C and (15)N CSA tensors are very sensitive to the protonation states in these compounds and, therefore, permit differentiating between the two ionization states, 4-aminopyrimidine and 4-aminopyrimidinium. Using density functional theory (DFT), we have calculated the magnetic shielding anisotropy tensors of C2, C6', and N4' and found excellent agreement between the computed and the experimental tensors. Our findings indicate that MAS NMR spectroscopy in conjunction with DFT calculations is a sensitive probe of ionization states in the thiamin cofactor. The results of this study will serve as a guide for characterization of ionization and tautomeric states of thiamin in complexes with thiamin-dependent enzymes.

Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic Angle Spinning NMR Spectroscopy.

Dynein light chain LC8 is the smallest subunit of the dynein motor complex and has been shown to play important roles in both dynein dependent and dynein independent physiological functions via its interaction with a number of its binding partners. It has also been linked to pathogenesis including roles in viral infections and tumorigenesis. Structural information for LC8-target proteins is critical to understanding the underlying function of LC8 in these complexes. However, some LC8-target interactions are not amenable for structural characterization by conventional structural biology techniques due to their large size, low solubility and crystallization difficulties. Here, we report magic angle spinning (MAS) NMR studies of the homodimeric apo-LC8 protein as a first effort in addressing more complex, multi-partner LC8-based protein assemblies. We have established site-specific backbone and side chain resonance assignments for the majority of the residues of LC8, and show TALOS+ predicted torsion angles ϕ and ψ in close agreement with most residues in the published LC8 crystal structure. Data obtained through these studies will provide the first step toward using MAS NMR to examine the LC8 structure, which will eventually be used to investigate protein-protein interactions in larger systems, which cannot be determined by conventional structural studies.

Determination of relative tensor orientations by γ-encoded chemical shift anisotropy/heteronuclear dipolar coupling 3D NMR spectroscopy in biological solids.

In this paper, we present 3D chemical shift anisotropy (CSA)/dipolar coupling correlation experiments, based on γ-encoded R-type symmetry sequences. The γ-encoded correlation spectra are exquisitely sensitive to the relative orientation of the CSA and dipolar tensors and can provide important structural and dynamic information in peptides and proteins. We show that the first-order (m = ±1) and second-order (m = ±2) Hamiltonians in the R-symmetry recoupling sequences give rise to different correlation patterns due to their different dependencies on the crystallite orientation. The relative orientation between CSA and dipolar tensors can be determined by fitting the corresponding correlation patterns. The orientation of (15)N CSA tensor in the quasi-molecular frame is determined by the relative Euler angles, α(NH) and ß(NH), when the combined symmetry schemes are applied for orientational studies of (1)H-(15)N dipolar and (15)N CSA tensors. The correlation experiments introduced here work at moderate magic angle spinning frequencies (10-20 kHz) and allow for simultaneous measurement of multiple sites of interest. We studied the orientational sensitivity of γ-encoded symmetry-based recoupling techniques numerically and experimentally. The results are demonstrated on [(15)N]-N-acetyl-valine (NAV) and N-formyl-Met-Leu-Phe (MLF) tripeptide.

Magic angle spinning NMR spectroscopy of thioredoxin reassemblies.

Differentially isotopically enriched 1-73((13)C,(15)N)/74-108((15)N) and 1-73((15)N)/74-108((13)C,(15)N) Escherichia coli thioredoxin reassemblies prepared by fragment complementation were investigated by high-resolution magic angle spinning solid-state NMR spectroscopy. Nearly complete resonance assignments, secondary and tertiary structure analysis are reported for 1-73((13)C,(15)N)/74-108((15)N) reassembled thioredoxin. Temperature dependence of the dipolar-assisted rotational resonance (DARR) spectra reveals the residues undergoing intermediate timescale motions at temperatures below - 15 degrees C. Analysis of the DARR intensity buildups as a function of mixing time in these reassemblies indicates that at long mixing times medium- and long-range cross-peaks do not experience dipolar truncation, suggesting that isotopic dilution is not required for gaining nontrivial distance restraints for structure calculations.