Protein sequential resonance assignments by combinatorial enumeration using 13C alpha chemical shifts and their (i,i-1) sequential connectivities

The need for the structural characterization of proteins on a genomic scale has brought with it demands for new technology to speed the structure determination process. In NMR, one bottleneck is the sequential assignment of backbone resonances. In this paper, we explore the computational complexity of the sequential assignment problem using only ¹³C chemical shift data and C (i,i–1) sequential connectivity information, all of which can potentially be obtained from a single three-dimensional NMR spectrum. Although it is generally believed that there is too much ambiguity in such data to provide sufficient information for sequential assignment, we show that a straightforward combinatorial search algorithm can be used to find correct and unambiguous sequential assignments in a reasonable amount of CPU time for small proteins (approximately 80 residues or smaller) when there is little missing data. The deleterious effect of missing or spurious peaks and the dependence on match tolerances is also explored. This simple algorithm could be used as part of a semi-automated, interactive assignment procedure, e.g., to test partial manually determined solutions fo uniqueness and to extend these solutions.     

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.     

Sequential resonance assignment of medium-sized15 N/13C-labeled proteins with projected 4D triple resonance NMR experiments

We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of ¹⁵N/¹³C-labeled proteins. A 3D ^{/ /}(CO)NHN experiment is recorded either in conjunction with 3D HNN< > or with the newly presented 3D HNN scheme. The first combination yields sequential assignments based on the measurement of¹³ C chemical shifts and provides a complete ¹H, ¹³C and ¹⁵N resonance assignment of polypeptide backbone and CH_n moieties. When employing the second combination, ¹³C=O chemical shifts are not measured, but the sequential assignment relies on both ¹³C and¹ H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized¹⁵ N/¹³C doubly labeled proteins.     

A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

The determination of the three-dimensional structure of a protein or the study of protein–ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p. Dedicated to Rüdiger Winter ( 06.04.2004)     

Solid-state NMR triple-resonance backbone assignments in a protein

Triple-resonance solid-state NMR spectroscopy is demonstrated to sequentially assign the 13C and 15N amide backbone resonances of adjacent residues in an oriented protein sample. The observed 13C chemical shift frequency provides an orientational constraint complementary to those measured from the 1H and 15N amide resonances in double-resonance experiments.     

A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: An application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C

A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30–100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni²⁺-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with ¹⁵N and/or¹³ C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.     

Bridging the gap: A set of selective 1H-15N-correlations to link sequential neighbors of prolines

Triple-resonance experiments are standard in the assignment of protein spectra. Conventional assignment strategies use ¹H-¹⁵N-correlations as a starting point and therefore have problems when proline appears in the amino acid sequence, which lacks a signal in these correlations. Here we present a set of amino acid selective pulse sequences which provide the information to link the amino acid on either side of proline residues and thus complete the sequential assignment. The experiments yield amino acid type selective ¹H-¹⁵N-correlations which contain signals from the amino protons of the residues either preceding or following proline in the amino acid sequence. These protons are correlated with their own nitrogen or with that of the proline. The new experiments are recorded as two-dimensional experiments and their performance is demonstrated by application to a 115-residue protein domain.     

Novel projected 4D triple resonance experiments for polypeptide backbone chemical shift assignment

Here we present a novel suite of projected 4D triple-resonance NMR experiments for efficient sequential assignment of polypeptide backbone chemical shifts in ¹³C/¹⁵N doubly labeled proteins. In the 3D HNN[CAHA] and 3D HNN(CO)[CAHA] experiments, the ¹³C and ¹H chemical shifts evolve in a common dimension and are simultaneously detected in quadrature. These experiments are particularly useful for the assignment of glycine-rich polypeptide segments. Appropriate setting of the ¹H radiofrequency carrier allows one to place cross peaks correlating either backbone ¹⁵N/¹H^N/¹³C or ¹⁵N/¹H^N/¹H chemical shifts in separate spectral regions. Hence, peak overlap is not increased when compared with the conventional 3D HNNCA and HNN(CA)HA. 3D HNN[CAHA] and 3D HNN(CO)[CAHA] are complemented by 3D reduced-dimensionality (RD) HNN COCA and HNN CACO, where ¹³C and ¹³C chemical shifts evolve in a common dimension. The ¹³C shift is detected in quadrature, which yields peak pairs encoding the ¹³C chemical shift in an in-phase splitting. This suite of four experiments promises to be of value for automated high-throughput NMR structure determination in structural genomics, where the requirement to independently sample many indirect dimensions in a large number of NMR experiments may prevent one from accurately adjusting NMR measurement times to spectrometer sensitivity.     

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].