A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H)-labeled proteins

Several HNCO-based three-dimensional experiments are described for the measurement of ¹³C(i–1)-¹³C(i–1), ¹⁵N(i)-¹³C(i–1), ¹⁵N(i)-¹³C(i), ¹⁵N(i)-¹³C(i–1), ¹H^N(i)-¹³C(i), ¹H^N(i)-¹³C(i–1), and ¹³C(i–1)-¹³C(i–1) scalar and dipolar couplings in ¹⁵N, ¹³C, (²H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superficially resembling an HNCO correlation spectrum, allowing accurate and simple measurement of couplings without introducing additional spectral crowding. Scalar and dipolar couplings are measured with good sensitivity from relatively large proteins, as demonstrated with three proteins: cardiac Troponin C, calerythrin and ubiquitin. Measurement of several dipolar couplings between spin-1/2 nuclei using spin-state selective 3D HNCO spectra provides a wealth of structural information.     

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between ¹H^N-¹³C, ¹⁵N-¹³C, ¹H^N-¹³C _i–1, ¹⁵N-¹³C _i–1, ¹H^N-¹³C_i–1, ¹⁵N-¹³C_i–1, and ¹³C_i–1-¹³C _i–1 without introducing nonessential spectral crowding compared with an ordinary two-dimensional ¹⁵N-¹H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of /-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure–activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.     

A method for incorporating dipolar couplings into structure calculations in cases of (near) axial symmetry of alignment

A method for incorporating dipolar coupling restraints into structure calculations is described which follows closely on methodology that has been recently presented for orienting peptide planes using dipolar couplings [Mueller et al. (2000) J. Mol. Biol., 300, 197–212] and is specifically developed for use in cases of an axially symmetric alignment tensor. Modeling studies on an all -helical protein, farnesyl diphosphate synthase, establish the utility of the approach. A global fold of the 370-residue maltose binding protein in complex with -cyclodextrin is obtained from experimentally derived restraints. The average pairwise rmsd values between the N- and C-terminal domains in this NMR structure and the corresponding regions in the X-ray structure of the protein are 2.8 and 3.1 Å, respectively.     

Structure-independent cross-validation between residual dipolar couplings originating from internal and external orienting media

Lanthanide-substituted calcium binding proteins are known to partially orient in high magnetic fields. Orientation provides residual dipolar couplings (rdc's). Two of these systems, Tm³⁺- and Dy³⁺-substituted calbindin D_9k, dissolved in an external orienting medium (nonionic liquid crystalline phase) provide rdc values which are the sum of those induced by the lanthanides and by the liquid crystalline phase on the native calcium binding protein. This structure-independent check shows the innocence of the orienting medium with respect to the structure of the protein in solution. Furthermore, the simultaneous use of lanthanide substitution and external orienting media provides a further effective tool to control and tune the orientation tensor.     

Residual dipolar coupling constants and structure determination of large DNA duplexes

Several NMR works have shown that long-range information provided by residual dipolar couplings (RDCs) significantly improve the global structure definition of RNAs and DNAs. Most of these are based on the use of a large set of RDCs, the collect of which requires samples labeled with ¹³C, ¹⁵N, and sometimes, ²H. Here, we carried out torsion-angle dynamics simulations on a non-self complementary DNA fragment of 17 base-pairs, d(GGAAAATATCTAGCAGT).(ACTGCTAGAGATTTTCC). This reproduces the U5 LTR distal end of the HIV-1 cDNA that contains the enzyme integrase binding site. Simulations aimed at evaluating the impact of RDCs on the structure definition of long oligonucleotides, were performed in incorporating (i) nOe-distances at both < 4.5 Å and < 5 Å; (ii) a small set of ¹³C-¹H RDCs, easily detectable at the natural abundance, and (iii) a larger set of RDCs only accessible through the ¹³C labeling of DNAs. Agreement between a target structure and a simulated structure was measured in terms of precision and accuracy. Results allowed to define conditions in which accurate DNA structures can be determined. We confirmed the strong impact of RDCs on the structure determination, and, above all, we found that a small set of RDC constraints (ca. 50) detectable at the natural abundance is sufficient to accurately derive the global and local DNA duplex structures when used in conjunction with nOe-distances < 5 Å.     

Induced alignment and measurement of dipolar couplings of an SH2 domain through direct binding with filamentous phage

Large residual ¹⁵N-¹H dipolar couplings have been measured in a Src homology II domain aligned at Pf1 bacteriophage concentrations an order of magnitude lower than used for induction of a similar degree of alignment of nucleic acids and highly acidic proteins. An increase in ¹ H and ¹⁵N protein linewidths and a decrease in T₂ and T₁ relaxation time constants implicates a binding interaction between the protein and phage as the mechanism of alignment. However, the associated increased linewidth does not preclude the accurate measurement of large dipolar couplings in the aligned protein. A good correlation is observed between measured dipolar couplings and predicted values based on the high resolution NMR structure of the SH2 domain. The observation of binding-induced protein alignment promises to broaden the scope of alignment techniques by extending their applicability to proteins that are able to interact weakly with the alignment medium.     

An HNCO-based Pulse Scheme for the Measurement of 13Cα-1Hα One-bond Dipolar couplings in 15N, 13C Labeled Proteins

A triple resonance pulse scheme is presented for recording 13C-1H one-bond dipolar couplings in 15N, 13C labeled proteins. HNCO correlation maps are generated where the carbonyl chemical shift is modulated by the 13C-1H coupling, with the two doublet components separated into individual data sets. The experiment makes use of recently described methodology whereby the protein of interest is dissolved in a dilute solution of bicelles which orient above a critical temperature, thus permitting measurement of significant couplings (Tjandra and Bax, 1997a). An application to the protein ubiquitin is described.     

Realistic Ensemble Models of Intrinsically Disordered Proteins Using a Structure-Encoding Coil Database

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Separated local field NMR experiments on oriented samples rotating at the magic angle

Biophysical studies on membrane proteins by solid state NMR (SSNMR) can be carried out directly in a membrane environment. Samples are usually prepared in form of multi-lamellar dispersions for magic angle sample spinning or as aligned multi-layers for orientation dependent NMR experiments without sample rotation. A new development is the application of MAS NMR to aligned samples (MAOSS; Magic Angle Oriented Sample Spinning). In combination with separated local field (SLF) experiments, size and orientation of heteronuclear dipolar couplings may be extracted from two-dimensional experiments which correlate dipolar couplings with isotropic chemical shifts. The orientation of these ¹H–X dipolar couplings can be directly related to the orientation of molecular groups in the sample. Here, we demonstrate the feasibility of these experiments on highly ordered polyethylene fibers which serve as model compound. Based on these data, the experiment is also applied to ordered multi-layers of bacteriorhodopsin (purple membrane) which is used as a model for aligned membrane proteins. We present a detailed analysis of different experimental designs with respect to angular sensitivity and the influence of residual sample disorder (“mosaic spread”). The results of the MAOSS-SLF experiment are discussed within the context of established solid state NMR experiments which are usually performed without sample rotation and we compare the data to orientation information obtained from X-ray diffraction.