Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B

Summary A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly ¹³C/¹⁵N-enriched proteins and can measure magnetization exchange rates in the 0.02 to >20s^–1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H resonances, followed by NOESY-or ROESY-type transfer to amide protons, which are dispersed by the amide ¹⁵N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H₂O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the central helix of the homologous protein calmodulin.     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Amide proton temperature coefficients as hydrogen bond indicators in proteins

Correlations between amide proton temperature coefficients (_HN/T) and hydrogen bonds were investigated for a data set of 793 amides derived from 14 proteins. For amide protons showing temperature gradients more positive than –4.6 ppb/K there is a hydrogen bond predictivity value exceeding 85%. It increases to over 93% for amides within the range between –4 and –1 ppb/K. Detailed analysis shows an inverse proportionality between amide proton temperature coefficients and hydrogen bond lengths. Furthermore, for hydrogen bonds of similar bond lengths, values of temperature gradients in -helices are on average 1 ppb/K more negative than in -sheets. In consequence, a number of amide protons in -helices involved in hydrogen bonds shorter than 2 Å show _HN/T < –4.6 ppb/K. Due to longer hydrogen bonds, 90% of amides in 3₁₀ helices and 98% in -turns have temperature coefficients more positive than –4.6ppb/K. Ring current effect also significantly influences temperature coefficients of amide protons. In seven out of eight cases non-hydrogen bonded amides strongly deshielded by neighboring aromatic rings show temperature coefficients more positive than –2 ppb/K. In general, amide proton temperature gradients do not change with pH unless they correspond to conformational changes. Three examples of pH dependent equilibrium showing hydrogen bond formation at higher pH were found. In conclusion, amide proton temperature coefficients offer an attractive and simple way to confirm existence of hydrogen bonds in NMR determined structures.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

Temperature coefficients of peptides dissolved in hexafluoroisopropanol monitor distortions of helices

Temperature coefficients are widely used as an indication of solvent accessibility to amide protons. Low temperature coefficients are related to low accessibility and are often interpreted as evidence for intramolecular hydrogen bonding. Conformational shifts, i.e. the difference between chemical shifts of a particular residue in a structured and in a random-coil conformation, provide information on secondary structure. In particular, negative CH conformational shifts are often used to delineate the extent of helical stretches. NH conformational shifts show large oscillations within a helix that have been interpreted as the result of helix distortions affecting hydrogen bond lengths. In the course of the study of different peptides that adopt a helical structure in the presence of the structure-inducing solvent hexafluoroisopropanol (HFIP), we have found a strong correlation between temperature coefficients and amide conformational shifts. However, contrary to the initial expectations, lower temperature coefficients were associated to amide protons involved in longer, and presumably weaker, hydrogen bonds. The correlation can be explained, however, assuming that, in helical peptides dissolved in HFIP, temperature affects the chemical shift of amide protons mainly by changing the average length of intramolecular hydrogen bonds and changes in solvent accessibility play only a secondary role under these experimental conditions. The pattern of temperature coefficients in helical peptides can therefore be used to identify short or long hydrogen bonds causing bending of the helix axis.     

Solvent Exchange Rates of Side-chain Amide Protons in Proteins

Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr. The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons. Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8. An NOE between a side-chain amide proton and a bound water molecule was also observed.     

Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N

Summary It is demonstrated that sequential resonance assignment of the backbone ¹H and ¹⁵N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate ¹H with the intraresidue ¹⁵N and with the ¹⁵N resonance of the next residue. The experiments are preferably conducted in D₂O, where very high resolution in the ¹⁵N dimension can be achieved by using ²H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in ¹³C and ¹⁵N. Complete backbone and ¹³C/¹H resonance assignments are presented.     

The effect of selective deuteration on magnetization transfer in larger proteins

Summary The effects of selective deuteration on calculated NOESY intensities have been analyzed for the structure of theE. coli trp aporepressor, a 25 kDa protein. It is shown that selectively deuteratedtrp aporepressor proteins display larger calculated NOESY intensities than those for the same interproton distances in the natural abundance protein. The relatively larger magnetization transfer is demonstrated by a comparison of the NOE build-up curves for specific proton pairs, and for the calculated NOE intensities of short-range NOEs to backbone amide protons. This increase in intensity is especially pronounced for the NH¹–NH¹⁺¹ cross peaks in the -helical regions, and particularly for amide protons of two sequential deuterated residues. The effect is shown to be further intensified for longer mixing times. It is also shown that in all cases, each amide proton exhibits stronger NOEs to its own side chain, with an enhanced effect for deuterated derivatives. This theoretical analysis demonstrates that an evaluation of the relative NOE intensities for different selectively deuterated analogs may be an important tool in assigning NMR spectra of large proteins. These results also serve as a guide for the interpretation of NOEs in terms of distances for structure calculations based on data using selectively deuterated proteins.     

The secondary structure of a pyrimidine-guanine sequence-specific ribonuclease possessing cytotoxic activity from the oocytes of Rana catesbeiana

Summary RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, ³J_NH and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel -sheets and three -helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 -protons located in the loop-1 region, an 2 helix, and three -strands (1, 3 and 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.Abbreviations DQF-COSY double-quantum-filtered correlation spectroscopy - DTT dithiothreitol - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - PE-1 N-terminal pyroglutamate - RC-RNase ribonuclease from the oocyte of Rana catesbeiana - TOCSY total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP sodium 3-trimethylsilylpropionate-2,2,3,3-d ₄     

Secondary structure determination by NMR spectroscopy of an immunoglobulin-like domain from the giant muscle protein titin

Summary We present the complete ¹⁵N and ¹H NMR assignment and the secondary structure of an immunoglobulin-like domain from the giant muscle protein titin. The assignment was obtained using homonuclear and ¹⁵N heteronuclear 2D and 3D experiments. The complementarity of 3D TOCSY-NOESY and 3D ¹⁵N NOESY-HSQC experiments, using WATERGATE for water suppression, allowed an efficient assignment of otherwise ambiguous cross peaks and was helpful in overcoming poor TOCSY transfer for some amino acids. The secondary structure is derived from specific NOEs between backbone - and amide protons, secondary chemical shifts of -protons and chemical exchange for the backbone amide protons. It consists of eight -strands, forming two -sheets with four strands each, similar to the classical -sandwich of the immunoglobulin superfamily, as previously predicted by sequence analysis. Two of the -strands are connected by type II -turns; the first -strand forms a -bulge. The whole topology is very similar to the only intracellular immunoglobulin-like domain for which a structure has been determined so far, i.e., telokin.     

Computer-assisted assignment of peptides with non-standard amino acids

Summary A comprehensive peptide assignment program and its application to a cyclic peptide, cyclosporin A, are presented in this paper. A group of graph theoretical algorithms using fuzzy logic are discussed with the aid of examples from cyclosporin A. The algorithms deal with heavily overlapped peaks, recover disjointed and distorted spin coupling networks, and include strategies for sequence-specific assignment. A procedure to extend the Protein Knowledge Base for automatically assigning non-standard amino acid residues is also presented. The program is capable of completely automated assignment for small peptides (20 residues). For such molecules, it is insensitive to whether the peptide chain is cyclic or acyclic, and to whether amide protons are present or absent. For larger peptides/proteins, more user interaction is required and the sequence-specific assignment step usually must proceed through fragments smaller than the full length to avoid problems due to occurrence of a combinatorial explosion. The program can be applied as a rigorous tool to check manual assignments. The fuzzy graph theoretical concepts built in the program are illustrated with 2D proton spectra of a peptide, but may be extended to higher-dimensional spectra, other biopolymers, natural products and other organic structures.     

Quantitative measurement of small through-hydrogen-bond and ‘through-space’1H-113Cd and1H-199Hg J couplings in metal-substituted rubredoxin fromPyrococcus furiosus

Summary A method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring¹H-¹¹³Cd and¹H-¹⁹⁹Hg J couplings in metal-substituted rubredoxin (M_r 5.4 kDa) fromPyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a through-space coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to¹⁹⁹Hg are significantly larger and fall in the 0.4–4 Hz range.     

High pressure NMR study of a small protein, gurmarin

The effect of pressure on the structure of gurmarin, a globular, 35-residue protein from Gymnema sylvestre, was studied in aqueous environment (95% 1H2O/5% 2H2O, pH 2.0) with an on-line variable pressure NMR system operating at 750 MHz. Two-dimensional TOCSY and NOESY spectra were measured as functions of pressure between 1 and 2000 bar at 40°C . Practically all the proton signals of gurmarin underwent some shifts with pressure, showing that the entire protein structure responds to, and is altered by, pressure. Most amide protons showed different degrees of low field shifts with pressure, namely 0–0.2 ppm with an average of 0.051 ppm at 2000 bar, showing that they are involved in hydrogen bonding and that these hydrogen bonds are shortened by pressure by different degrees. The tendency was also confirmed that the chemical shifts of the amide protons exposed to the solvent (water) are more sensitive to pressure than those internally hydrogen bonded with carbonyls. The pressure-induced shifts of the H signals of the residues in the -sheet showed a negative correlation with the folding shifts (difference between the shift at 1 bar and that of a random coil), suggesting that the main-chain torsion angles of the -sheet are slightly altered by pressure. Significant pressure-induced shifts were also observed for the side-chain protons (but no larger than 10% of the folding shifts), demonstrating that the tertiary structure of gurmarin is also affected by pressure. Finally, the linearity of the pressure-induced shifts suggests that the compressibility of gurmarin is invariant in the pressure range between 1 and 2000 bar.     

A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30–40 kDa to overcome problems arising from rapid ¹H and ¹³C transverse relaxation. However, ¹H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D₂O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H₂O. As a side-effect partial C protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary ¹H^N- and ¹H-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D₂O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of C protonation of individual amino acids is determined quantitatively and transverse relaxation properties of ¹H^N and ¹⁵N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.     

Phosphorylation mechanisms in chemical evolution

An objective of this work is to elucidate the mechanism of phosphorylation of nucleosides in amide solvents and in urea. A second objective is to assess the importance of phosphorylation and dephosphorylation of nucleotide derivatives in amide environments. Although the most complex amide studied here was N-methylacetamide, inferences are made on the importance of dephosphorylation for nucleotides in oligopeptide environments.Phosphorylations in amide solvents and in urea are suggested to proceed through monomeric metaphosphate, which was first postulated as a reaction intermediate thirty years ago (Butcher and Westheimer, 1955). Phosphorylation of nucleosides and nucleotides and dephosphorylation of nucleotide derivatives have been studied in formamide, N-methylformamide, urea and N-methylacetamide. Hydrated forms of 5-ADP and 5ATP are unstable in hot amide solvents and in urea. They decompose to a mixture of adenosine and its phosphorylated derivatives. The rate of decomposition is much slower in N-methylacetamide than in formamide or urea. Experiments designed to prepare oligonucleotides in the presence of oligopeptides have been reported (White, 1983). According to the present study, it is not unreasonable to expect that nucleotide derivatives can be condensed with nucleosides to form oligonucleotides in a peptide environment. However, nucleotide monomers such as 5-ATP, 5-ADP or 5AMP will suffer isomerization or decomposition during condensation use of activated phosphate derivatives is preferable.Monomeric metaphosphate has not been isolated or characterized in amide solvents. It is proposed here as a reaction intermediate, probably in a complexed form with the amide.     

Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide

Summary We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with ¹⁵N. The requirement of ¹³C-/¹⁵N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., _0c~1). The technique was applied to an unstructured ¹⁵N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s^-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.     

Unambiguous NOE assignments in proteins by a combination of through-bond and through-space correlations

Heteronuclear editing has found widespread use in the detection ofproton–proton dipolar interactions in isotopically labelled proteins.However, in cases where both the resonances of protons and directly bound¹³C or ¹⁵N spins of two or more sites aredegenerate, unambiguous assignments are difficult to obtain by conventionalmethods. Here, we present simple extensions of well-known triple-resonancepulse sequences which improve the dispersion of NOESY spectra. In order torecord the chemical shifts of backbone nuclei which allow a resolution ofoverlapping cross peaks, the magnetization is relayed via the scalarcoupling network either before or after the NOE mixing period. The novelpulse sequences are applied to flavodoxin from the sulfate-reducing organismDesulfovibrio vulgaris. A number of previously unassigned NOE interactionsinvolving -, - and amide protons can be unequivocallyidentified, suggesting that the accuracy of protein structure determinationcan be improved.