A systematic analysis of backbone amide assignments achieved via combinatorial selective labelling of amino acids

With the advent of high-yield cell-free expressions systems, many researchers are exploiting selective isotope labelling of amino acids to increase the efficiency and accuracy of the NMR assignment process. We developed recently a combinatorial selective labelling (CSL) method capable of yielding large numbers of residue-type and sequence-specific backbone amide assignments, which involves comparing cross-peak intensities in ¹H–¹⁵N HSQC and 2D ¹H–¹⁵N HNCO spectra collected for five samples containing different combinations of ¹³C- and ¹⁵N-labelled amino acids [Parker MJ, Aulton-Jones M, Hounslow A, Craven C J (2004) J Am Chem Soc 126:5020–5021]. In this paper we develop a robust method for establishing the reliability of these assignments. We have performed a detailed statistical analysis of the CSL data collected for a model system (the B1 domain of protein G from Streptococcus), developing a scoring method which allows the confidence in assignments to be assessed, and which enables the effects of overlap on assignment fidelity to be predicted. To further test the scoring method and also to assess the performance of CSL in relation to sample quality, we have applied the method to the CSL data collected for GFP in our previous study.     

1H, 15N and 13C assignments of yellow fluorescent protein (YFP) Venus

We present here the backbone and side-chain NMR assignments of YFP Venus, a 238-residue protein that emits yellow fluorescence in its native state. Venus is a variant of the green fluorescent protein (GFP), which has improved chromophore maturation and brightness, and the photochemistry and photophysics of which are insensitive to experimental conditions, such as the pH value and buffer content, making it a favourable biomarker.     

Generation and relaxation of high rank coherences in AX<Subscript>3</Subscript> systems in a selectively methionine labelled SH2 domain

The usefulness of selective isotope labelling patterns is demonstrated using the C-terminal SH2 domain of PLC-γ1 selectively ¹³C labelled at methionine methyl groups. We demonstrate the generation and relaxation of coherences that are second rank in protons and first rank in carbons that derive from quadrupolar order in protons. The decay rates of second rank double quantum proton coherences are measured. These terms exhibit fewer channels for cross-correlated relaxation compared to single quantum coherences. Our results indicate the potential application of the measurement of high order proton coherences to the analysis of dynamics in methyl-bearing side chains.     

APSY-NMR with proteins: practical aspects and backbone assignment

Automated projection spectroscopy (APSY) is an NMR technique for the recording of discrete sets of projection spectra from higher-dimensional NMR experiments, with automatic identification of the multidimensional chemical shift correlations by the dedicated algorithm GAPRO. This paper presents technical details for optimizing the set-up and the analysis of APSY-NMR experiments with proteins. Since experience so far indicates that the sensitivity for signal detection may become the principal limiting factor for applications with larger proteins or more dilute samples, we performed an APSY-NMR experiment at the limit of sensitivity, and then investigated the effects of varying selected experimental parameters. To obtain the desired reference data, a 4D APSY-HNCOCA experiment with a 12-kDa protein was recorded in 13 min. Based on the analysis of this data set and on general considerations, expressions for the sensitivity of APSY-NMR experiments have been generated to guide the selection of the projection angles, the calculation of the sweep widths, and the choice of other acquisition and processing parameters. In addition, a new peak picking routine and a new validation tool for the final result of the GAPRO spectral analysis are introduced. In continuation of previous reports on the use of APSY-NMR for sequence-specific resonance assignment of proteins, we present the results of a systematic search for suitable combinations of a minimal number of four- and five-dimensional APSY-NMR experiments that can provide the input for algorithms that generate automated protein backbone assignments.     

1H-filtered correlation experiments for assignment and determination of coupling constants in backbone labelled proteins

The implementation of [¹³C,¹³C,¹⁵N,²H] labelled amino acids into proteins allows the acquisition of high resolution triple resonance experiments. We present for the first time resonance assignments facilitated by this new labelling strategy. The absence of ¹J_C,C couplings enables us to measure ¹J_C,C scalar and ¹D_C,C residual dipolar coupling constants using modified HNCA experiments which do not suffer from sensitivity losses characteristic for ¹³C constant time experiments.     

Improved 4D NMR experiments for the assignment of backbone nuclei in13C/15N labelled proteins

Summary We recently proposed a novel 4D NMR strategy for the assignment of backbone nuclei in¹³C/¹⁵N-labelled proteins (Boucher et al., 1992). Intra-residue (and many sequential) assignments are obtained from a HCANNH experiment, whereas sequential assignments are based on a complementary HCA(CO)NNH experiment. We present here new constant time 4D HCANNH, HCA(CO)NNH and HNCAHA experiments that are more sensitive. Some of the data were presented at the 33rd ENC held at Asilomar, California, U.S.A., in April 1992.     

Statistical analysis of double NOE transfer pathways in proteins as measured in 3D NOE-NOE spectroscopy

Summary The recent development of three-dimensional NMR spectroscopy has alleviated the problem of overlap of resonances. However, also for the 3D experiments resonance assignment strategies have usually relied upon knowledge about spin systems, combined with information about short (sequential) distances. For doubly (¹⁵N/¹³C)-labelled molecules, a novel assignment strategy has been developed. In this paper we address the possibilities of an assignment strategy for proteins, based solely upon the use of NOE data. For this, the 3D NOE-NOE experiment seems most suitable. Therefore, we have made a theoretical evaluation of double NOE transfer pathways in 28 protein crystal structures. We identify 95 connectivities which are most likely to be observed as cross peaks in a 3D NOE-NOE spectrum of a protein. Given the occurrence of one of these 95 connectivities, we evaluate the chances of occurrence for the others. Analysis of these conditional probabilities allowed the construction of five patterns of related, highly correlated cross peaks which resemble the conventional idea of spin systems to some extent and may provide a basis for assignment and secondary structure analysis from 3D NOE-NOE data alone.Dedicated to the memory of Professor V.F. Bystrov     

Two- and three-dimensional 31P-driven NMR procedures for complete assignment of backbone resonances in oligodeoxyribonucleotides

Summary We describe a strategy for sequential assignment of ³¹P and deoxyribose ¹H NMR resonances in oligodeoxyribonucleotides. The approach is based on ³¹P–¹H J-cross-polarization (hetero TOCSY) experiments, recently demonstrated for the assignment of resonances in RNA [Kellogg, G.W. (1992) J. Magn. Reson., 98, 176; Kellogg, G.W. et al. (1992) J. Am. Chem. Soc., 114, 2727]. Two-dimensional hetero TOCSY and hetero TOCSY-NOESY experiments are used to connect proton spin systems from adjacent nucleotides in the dodecamer d(CGCGAATTCGCG)₂ entirely on the basis of through-bond scalar connectivities. All phosphorus resonances of the dodecamer are assigned by this method, and many deoxyribose ¹H resonances can be assigned as well. A new three-dimensional hetero TOCSY-NOESY experiment is used for backbone proton 4, 5 and 5 resonance assignments, completing assignments begun on this molecule in 1983 [Hare, D.R. et al. (1983) J. Mol. Biol., 171, 319]. Numerical simulations of the time dependence of coherence transfer aid in the interpretation of hetero TOCSY spectra of oligonucleotides and address the dependence of hetero TOCSY and related spectra on structural features of nucleic acids. The possibility of a generalized backbone-driven ¹H and ³¹P resonance-assignment strategy for oligonucleotides is discussed.To whom correspondence should be addressed.     

Selective labelling of phosphorylase kinase with fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) is a highly specific inhibitor of rabbit muscle phosphorylase kinase. The rapid inhibition process is accompanied by an almost exclusive incorporation of fluorescein into the α sub-unit. A molar ratio of 0.8 mol FITC per mol α subunit for a 60% inhibited kinase was calculated. Mg²⁺ and Mg²⁺-ATP completely block the inhibitory effect of FITC, but ATP, ADP and Ca²⁺ have no significant effect on FITC inhibition. Trypsin-activated phosphorylase kinase is not inactivated by FITC, while the fluorescein-modified enzyme can be activated by digestion with trypsin to the same level of activity of trypsin-activated unmodified enzyme.     

Selective chemical depolymerization of rhamnogalacturonans

A method was developed to selectively methyl esterify and then cleave GalA residues in pectic polysaccharides. The method was optimized using a rhamnogalacturonan (RG) from Arabidopsis mucilage as a model compound. The carboxyl group of the GalA residues in the RG was selectively methyl esterified using tetrabutylammonium fluoride and iodomethane in Me(2)SO containing 8% water. A 1D HMQC NMR method to determine the degree of methyl esterification was developed using (13)C-iodomethane as the methylating agent. The methyl-esterified pectins were fragmented by beta-elimination in 0.2M sodium borate, pH7.3, at 125 degrees C. The resulting oligoglycosyl fragments, which contain a nonreducing 4-deoxy-beta-l-threo-hex-4-enepyranosyluronic acid residue, were characterized using MALDI-TOF mass spectrometry, monosaccharide composition analysis, and 1D and 2D (1)H and (13)C NMR spectroscopy. Application of this method to branched RG from potato generated low-molecular-weight fragments containing two residues from the RG backbone and a single side chain. In contrast, the fragments obtained when RG is treated with RG lyase contain a minimum of four backbone residues. The chemical method thus facilitates the release and structural characterization of the side-chain structures of RG obtained from various plant sources. The method also provides a convenient method for generating fully or partially methyl-esterified homogalacturonans.     

Metabolic differentiation of Arabidopsis treated with methyl jasmonate using nuclear magnetic resonance spectroscopy

NMR spectroscopy combined with principal component analysis was applied to Arabidopsis thaliana treated with methyl jasmonate in order to obtain macroscopic metabolic changes caused by the treatment. As the first step several chromatographic and NMR spectroscopic techniques were utilized to identify metabolites of Arabidopsis. Sephadex LH-20 showed a high efficiency in the separation of phenolic metabolites in the plant. For identification of minor metabolites two-dimensional J-resolved NMR technique was directly applied to the plant extract and results in a number of elucidation of the metabolites of which signals overlap in ¹H NMR spectra. The chemical structure of the identified metabolites were confirmed by various two-dimensional NMR spectroscopy including correlated spectroscopy, heteronuclear single quantum coherence, and heternuclear multiple bond correlation. As next step, a statistical approach, principal component analysis based on projected J-resolved NMR spectra was performed for metabolic alteration of methyl jasmonate-treated Arabidopsis. The results show that methyl jasmonate caused an increase of flavonoids, fumaric acid, sinapoyl malate, sinigrin, tryptophan, valine, threonine, and alanine and a decrease of malic acid, feruloyl malate, glutamine, and carbohydrates after 24 h treatment.     

A new triple-resonance experiment for the sequential assignment of backbone resonances in proteins

Summary A new protocol is described for obtaining intraresidual and sequential correlations between carbonyl carbons and amide ¹H and ¹⁵N resonances of amino acids. Frequency labeling of ¹³CO spins occurs during a period required for the ¹³C-¹⁵N polarization transfer, leading to an optimized transfer efficiency. In a four-dimensional version of the experiment, ¹³C chemical shifts are used to improve the dispersion of signals. The resonance frequencies of all backbone nuclei can be detected in a 3D variant in which cross peaks are split along two frequency axes. This pulse scheme is the equivalent of a five-dimensional experiment. The novel pulse sequences are applied to flavodoxin from Desulfovibrio vulgaris.     

Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.     

Selective metabolic labelling of intracellular parasite proteins using ricin

The characterization of protein synthesis by eukaryotic intracellular parasites is inherently difficult because it may represent only a small fraction of host cell protein synthesis. Here, Mark Carrington, Yael Shochat and Anne Gurnett describe a method for overcoming this problem through the use of the toxin ricin to inhibit host cell protein synthesis specifically.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

3D 13C-15N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13C-15N-double-labeled proteins

Summary The pulse sequence of a new constant-time 3D triple-resonance experiment, ct-HA[CAN]HN, is presented. This experiment delineates exclusively scalar connectivities and uses ¹³C–¹⁵N heteronuclear two-spin coherence to overlay the chemical shift evolution periods of the ¹³C and ¹⁵N nuclei, thereby providing the four resonance frequencies of the -proton, the -carbon, the amide nitrogen, and the amide proton of a given amino acid residue in three dimensions. This experiment promises to be a valid alternative to 4D experiments, providing the same information on intraresidue polypeptide backbone connectivities in ¹³C-¹⁵N-double-labeled proteins.Abbreviations 3D, 4D three-dimensional, four-dimensional - TPPI time-proportional phase incrementation - ct constant-time - rf radiofrequency - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - glutaredoxin(C14S) mutant E. coli glutaredoxin with the cysteine at position 14 replaced by serine     

Computer assignment of the backbone resonances of labelled proteins using two-dimensional correlation experiments

Summary We present ALPS (Assignment for Labelled Protein Spectra), a flexible computer program for the automatic assignment of backbone NMR resonances of ¹⁵N/¹³C-labelled proteins. The program constructs pseudoresidues from peak-picking lists of a set of two-dimensional triple resonance experiments and uses either a systematic search or a simulated annealing-based optimization to perform the assignment. This method has been successfully tested on two-dimensional triple resonance spectra of Rhodobacter capsulatus ferrocytochrome c ₂ (116 amino acids).     

Mars - robust automatic backbone assignment of proteins

MARS a program for robust automatic backbone assignment of (13)C/(15)N labeled proteins is presented. MARS does not require tight thresholds for establishing sequential connectivity or detailed adjustment of these thresholds and it can work with a wide variety of NMR experiments. Using only (13)C(alpha)/(13)C(beta) connectivity information, MARS allows automatic, error-free assignment of 96% of the 370-residue maltose-binding protein. MARS can successfully be used when data are missing for a substantial portion of residues or for proteins with very high chemical shift degeneracy such as partially or fully unfolded proteins. Other sources of information, such as residue specific information or known assignments from a homologues protein, can be included into the assignment process. MARS exports its result in SPARKY format. This allows visual validation and integration of automated and manual assignment.     

Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling

Paramagnetic relaxation has been used to monitor the formation of structure in the folding peptide chain of guanidinium chloride-denatured acyl-coenzyme A-binding protein. The spin label (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSL) was covalently bound to a single cysteine residue introduced into five different positions in the amino acid sequence. It was shown that the formation of structure in the folding peptide chain at conditions where 95% of the sample is unfolded brings the relaxation probe close to a wide range of residues in the peptide chain, which are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded state, and that these may be of importance to the initiation of protein folding.