Validating the use of database potentials in protein structure determination by NMR

The refinement of protein structures determined by nuclear magnetic resonance spectroscopy against database potentials of mean force allows for the exclusion of unfavourable conformations of the protein backbone during a structure calculation, resulting in protein structures with a marked improvement in Ramachandran statistics. In this communication, we use multiple sets of residual dipolar couplings as quality assessment criteria for several proteins and show that not only do the Ramachandran and structural quality statistics improve, but a significant improvement in the accuracy of structures is achieved upon refinement.     

Role of scalar protons in metabolic energy generation in lactic acid bacteria

Lactic acid bacteria are able to generate a protonmotive force across the cytoplasmic membrane by various metabolic conversions without involvement of substrate level phosphorylation or proton pump activity. Weak acids like malate and citrate are taken up in an electrogenic process in which net negative charge is translocated into the cell thereby generating a membrane potential. The uptake is either an exchange process with a metabolic end-product (precursor/ product exchange) or a uniporter mechanism. Subsequent metabolism of the internalized substrate drives uptake and results in the generation of a pH gradient due to the consumption of scalar protons. The generation of the membrane potential and the pH gradient involve separate steps in the pathway. Here it is shown that they are nevertheless coupled. Analysis of the pH gradient that is formed during malolactic fermentation and citrate fermentation shows that a pH gradient, inside alkaline, is formed only when the uptake system forms a membrane potential, inside negative. These secondary metabolic energy generating systems form a pmf that consists of both a membrane potential and a pH gradient, just like primary proton pumps do. It is concluded that the generation of a pH gradient, inside alkaline, upon the addition of a weak acid to cells is diagnostic for an electrogenic uptake mechanism translocating negative charge with the weak acid.     

Interference between Cross-correlated Relaxation and the Measurement of Scalar and Dipolar Couplings by Quantitative J

The effects of cross-correlated relaxation in Quantitative J methods are analyzed. One-bond ¹H–¹³C scalar and dipolar couplings of protein methine and methylene sites are obtained by monitoring proton and carbon magnetization in Quantitative J experiments. We find that scalar and dipolar couplings of the same pair of nuclei vary depending on the type of magnetization involved. These discrepancies can be as large as several Hz for methylene moieties. The contribution of dynamic frequency shifts, which are known to affect J couplings, is too small to explain the observed differences. We show that processes of magnetization transfer originated by cross-correlated relaxation are largely responsible for these discrepancies. We estimate the error transferred to methylene J values by cross-correlation interference, and show that is close to the experimentally observed one. Furthermore, this analysis indicates that cross-correlated relaxation effects under isotropic and anisotropic media differ, indicating that errors are not cancelled in residual dipolar coupling measurements.     

NMR Structure of the N-Terminal Domain of Capsid Protein from the Mason-Pfizer Monkey Virus

The high-resolution structure of the N-terminal domain (NTD) of the retroviral capsid protein (CA) of Mason-Pfizer monkey virus (M-PMV), a member of the betaretrovirus family, has been determined by NMR. The M-PMV NTD CA structure is similar to the other retroviral capsid structures and is characterized by a six α-helix bundle and an N-terminal β-hairpin, stabilized by an interaction of highly conserved residues, Pro1 and Asp57. Since the role of the β-hairpin has been shown to be critical for formation of infectious viral core, we also investigated the functional role of M-PMV β-hairpin in two mutants (i.e., ΔP1NTDCA and D57ANTDCA) where the salt bridge stabilizing the wild-type structure was disrupted. NMR data obtained for these mutants were compared with those obtained for the wild type. The main structural changes were observed within the β-hairpin structure; within helices 2, 3, and 5; and in the loop connecting helices 2 and 3. This observation is supported by biochemical data showing different cleavage patterns of the wild-type and the mutated capsid-nucleocapsid fusion protein (CANC) by M-PMV protease. Despite these structural changes, the mutants with disrupted salt bridge are still able to assemble into immature, spherical particles. This confirms that the mutual interaction and topology within the β-hairpin and helix 3 might correlate with the changes in interaction between immature and mature lattices.     

Pulse schemes for the measurement of3 JC′Cγ and3 JNCγ scalar couplings in 15N,13C uniformly labeled proteins

Pulse sequences are presented for the measurement of³J_CC and³J_NC scalar couplings for allC containing residues in¹⁵N,¹³C uniformly labeled proteins. The methodsdescribed are based on quantitative J correlation spectroscopy pioneered byBax and co-workers [Bax et al. (1994) Methods Enzymol., 239, 79–105].The combination of ³J_CC and³J_NC scalar coupling constants allows theassignment of discrete rotameric states about the ₁ torsion angle in cases where such states exist or, alternatively,facilitates the establishment of noncanonical ₁conformations or the presence of rotameric averaging. The methods areapplied to a 1.5 mM sample of staphylococcal nuclease.     

Application of dipolar coupling data to the refinement of the solution structure of the sarcin-ricin loop RNA

Residual dipolar couplings can provide the long-range information that most NMR solution structures lack. The use of such data in protein structure determinations is now fairly routine, but even though these data should be much more useful for nucleic acids, their application to nucleic acid structure determination is still in its infancy. Here we present a method for producing accurate, dipolar-refined structures of nucleic acids that is more efficient than those used previously, and apply it to E73, a 29 nucleotide RNA that includes the sarcin-ricin loop from rat 28S rRNA. The results enable us to address the differences between the crystal structure of E73 and the solution structure proposed for it previously.     

Refined NMR structure of α-sarcin by 15N–1H residual dipolar couplings

¹⁵N–¹H residual dipolar couplings (RDC) have been used as additional restraints to refine the solution structure of the ribotoxin -sarcin. The RDC values were obtained by partial alignment of -sarcin in the binary mixture of n-dodecyl hexa(ethylene glycol)/hexanol. A total of 131 RDCs were measured and 106 were introduced in the final steps of the calculation protocol following the main calculation based on nuclear Overhauser enhancements and torsion angle restraints. A homogeneous family of 81 conformers was obtained. The resulting average pairwise root-mean-square deviation corresponding to the superposition of the 20 best structures is 0.69±0.12 Å for the backbone and 1.29±0.14 Å for all heavy atoms. The new structural features derived from the refined structure, compared with the non-refined structure of -sarcin, consist of new hydrogen bonds and a better definition of the backbone conformation. In particular, the loop segment spanning Gly 60 to Lys 70 shows a single conformation, corresponding to the most populated family of conformers observed in the unrefined structure. The information derived from the analysis of the refined structure and the comparison with the homologous protein restrictocin could help in establishing further structure–function relationships concerning -sarcin which can be reasonably extrapolated to other members of the ribotoxin family.     

Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cgamma-Hgamma/Cbeta-Hbeta residual dipolar couplings: application to the 34-kDa enzyme IIA(chitobiose)

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a ¹⁵N/¹³C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)–H_N(i) and Hγ(i)–H_N(i+1) NOEs in a 3D ¹⁵N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine ¹³C–¹H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA^Chitobiose, a 34 kDa homotrimeric phosphotransferase protein.     

Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment

Novel NMR pulse schemes for simultaneous measurement of ¹ D _CHand ² D _NHresidual dipolar couplings in proteins is presented. We show that ² D _NHcoupling can be very useful for protein structure determination. The ² D _NHcoupling can be measured from ¹⁵N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large ¹ D _CHcoupling. The dynamic range of ² D _NHcoupling is comparable to ¹ D _NC coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on ¹⁵N/¹³C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both ¹ D _CHand ² D _NHcouplings, was obtained.