Arthur Pardi

Calibration of the angular dependence of the amide proton-Cα proton coupling constants, 3JHNα, in a globular protein: use of 3JHnα for identification of helical secondary structure

Abstract The vicinal amide proton-C α proton spin-spin coupling constants, 3 J HNα in the globular protein basic pancreatic trypsin inhibitor (BPTI) have been measured using phase-sensitive correlated spectroscopy at high digital resolution. In conjunction with the crystal structure of BPTI, these data were used to calibrate the correlation between 3 J HN α and the dihedral angle φ. The resulting “BPTI curve” is 3 J HN α = 6.4 cos 2 θ − 1.4 cos θ + 1.9 (θ = ¦φ − 60° ¦) . It is further shown that measurement of the spin-spin couplings 3 J HN α presents an independent, reliable method for identification of the location of helical structure in the amino acid sequence of proteins.

Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions

Dipolar coupling interactions represent an extremely valuable source of long-range distance and angle information that was previously not available for solution structure determinations of macromolecules. This is because observation of these dipolar coupling data requires creating an anisotropic environment for the macromolecule. Here we introduce a new method for generating tunable degrees of alignment of macromolecules by addition of magnetically aligned Pf1 filamentous bacteriophage as a cosolute. This phage-induced alignment technique has been used to study 1H-1H, 1H-13C, and 1H-15N dipolar coupling interactions in a DNA duplex, an RNA hairpin and several proteins including thioredoxin and apo-calmodulin. The phage allow alignment of macromolecules over a wide range of temperature and solution conditions and thus represent a stable versatile method for generating partially aligned macromolecules in solution.

[15] Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions

Publisher Summary This chapter describes the application of filamentous bacteriophage as a versatile tool for generating partially ordered solutions of nucleic acids and proteins. The Pfl phage system described in the chapter is an ideal method for obtaining a tunable degree of alignment of RNA and DNA oligomers and some proteins in solution, which then allows the measurement of homonuclear and heteronuclear dipolar coupling interactions. The Pfl filamentous phage system is ideally suited for obtaining a tunable degree of alignment in RNA and DNA molecules and acidic proteins for the observation of dipolar couplings. The phage particles are fully aligned at magnetic fields as low as 300 MHz and are stable indefinitely at a range of temperatures and buffer conditions. These dipolar couplings provide long-range structural information that is presently unavailable by standard solution nuclear magnetic resonance (NMR) techniques; therefore, in the future, dipolar coupling data may prove to be as indispensable as 1 H– 1 H nuclear Overhauser effect (NOE) in the solution structure determinations of macromolecules. This should be especially true for structure determinations of RNA and DNA oligomers where the low density of protons compared to proteins leads to fewer useful NOE distance constraints, and therefore generally less well-defined structures. In addition, the current solution NMR techniques are generally unable to determine global structural features, such as bending in nucleic acids, due because of the local nature of the NOE distance constraints.

NMR Methods for Studying the Structure and Dynamics of RNA

Proper functioning of RNAs requires the formation of complex three‐dimensional structures combined with the ability to rapidly interconvert between multiple functional states. This review covers recent advances in isotope‐labeling strategies and NMR experimental approaches that have promise for facilitating solution structure determinations and dynamics studies of biologically active RNAs. Improved methods for the production of isotopically labeled RNAs combined with new multidimensional heteronuclear NMR experiments make it possible to dramatically reduce spectral crowding and simplify resonance assignments for RNAs. Several novel applications of experiments that directly detect hydrogen‐bonding interactions are discussed. These studies demonstrate how NMR spectroscopy can be used to distinguish between possible secondary structures and identify mechanisms of ligand binding in RNAs. A variety of recently developed methods for measuring base and sugar residual dipolar couplings are described. NMR residual dipolar coupling techniques provide valuable data for determining the long‐range structure and orientation of helical regions in RNAs. A number of studies are also presented where residual dipolar coupling constraints are used to determine the global structure and dynamics of RNAs. NMR relaxation data can be used to probe the dynamics of macromolecules in solution. The power dependence of transverse rotating‐frame relaxation rates was used here to study dynamics in the minimal hammerhead ribozyme. Improved methods for isotopically labeling RNAs combined with new types of structural data obtained from a growing repertoire of NMR experiments are facilitating structural and dynamic studies of larger RNAs.

The cavity-chaperone Skp protects its substrate from aggregation but allows independent folding of substrate domains

Outer membrane proteins (OMPs) of Gram-negative bacteria are synthesized in the cytosol and must cross the periplasm before insertion into the outer membrane. The 17-kDa protein (Skp) is a periplasmic chaperone that assists the folding and insertion of many OMPs, including OmpA, a model OMP with a membrane embedded β-barrel domain and a periplasmic αβ domain. Structurally, Skp belongs to a family of cavity-containing chaperones that bind their substrates in the cavity, protecting them from aggregation. However, some substrates, such as OmpA, exceed the capacity of the chaperone cavity, posing a mechanistic challenge. Here, we provide direct NMR evidence that, while bound to Skp, the β-barrel domain of OmpA is maintained in an unfolded state, whereas the periplasmic domain is folded in its native conformation. Complementary cross-linking and NMR relaxation experiments show that the OmpA β-barrel is bound deep within the Skp cavity, whereas the folded periplasmic domain protrudes outside of the cavity where it tumbles independently from the rest of the complex. This domain-based chaperoning mechanism allows the transport of β-barrels across the periplasm in an unfolded state, which may be important for efficient insertion into the outer membrane.

Solution structures of the rabbit neutrophil defensin NP-5.

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.

Nuclear magnetic resonance studies of the hammerhead ribozyme domain: Secondary structure formation and magnesium ion dependence☆

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1:1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C.G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.

Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution.

Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein-nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA-ligand titration experiments, measurement of residual dipolar (15)N-(1)H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*(GA) RNA kissing complex and a 26 kDa tRNA.

Refined Solution Structure of the Iron-responsive Element RNA Using Residual Dipolar Couplings

The iron-responsive element (IRE) is a 30nt RNA motif located in the non-coding regions of mRNAs of proteins involved in iron regulation. In humans, the IRE plays a direct role in the control of iron levels by post-transcriptional regulation of the ferritin and transferrin receptor proteins through highly specific recognition by IRE-binding proteins. The IRE fold is representative of many RNA motifs that contain helical domains separated by a bulge or internal loop. The global structures of such extended multi-domain RNAs are not well defined by conventional NMR-distance and torsion angle structural restraints. Residual dipolar couplings (RDCs) are employed here to better define the global structure of the IRE RNA in solution. RDCs contain valuable long-range structural information that compliments the short-range structural data derived from standard NOE-distance and torsion angle restraints. Several approaches for estimating alignment tensor parameters and incorporating RDCs into RNA structure determinations are compared. Both the local and global structure of the IRE are improved significantly by refinement with RDCs. These RDC refinements provide insight on the conformational dynamics of the IRE. These studies highlight some issues that need to be addressed when incorporating RDCs in solution structure determinations of nucleic acids. The approach used here should prove valuable for structure determinations of various multi-domain systems.

NMR chemical exchange as a probe for ligand-binding kinetics in a theophylline-binding RNA aptamer.

The apparent on and off rate constants for binding of theophylline to its RNA aptamer in the absence of Mg(2+) were determined here by 2D (1)H-(1)H ZZ-exchange NMR spectroscopy. Analysis of the buildup rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent k(on) of 600 M(-1) s(-1). This small apparent k(on) results because the free RNA exist as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data found here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg(2+). The kinetic data found here also explain a very unusual property of this RNA-theophylline system: slow exchange on the NMR chemical shift time scale for a weakly binding complex. To our knowledge, it is unprecedented to have such a weakly binding complex (K(d) approximately 3.0 mM at 15 degrees C) show slow exchange on the NMR chemical shift time scale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small k(on). Comparisons with other ligand-receptor interactions are presented.