Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D₂O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d₁₀. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.     

Use of biosynthetic fractional 13C-labeling for backbone NMR assignment of proteins

We describe a simple approach to classify amino acid residue types in NMR spectra of proteins for supporting the backbone resonance assignments. It makes use of the differences in biosynthetic pathways of the 20 amino acids in Escherichia coli. Therefore, it is distinct from the parameters routinely exploited in the backbone resonance assignment such as chemical shifts and spin topology information. The combination of biosynthetically directed fractional 13C-labeling and uniform 15N-labeling enables us to obtain both residue-type specific information and sequential connectivities from a single protein sample. The residue-type classification exploiting biosynthetic pathways can be used for accelerating the conventional backbone assignment procedure.     

Biosynthetically directed 2H labelling for stereospecific resonance assignments of glycine methylene groups

Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific resonance assignments can readily be obtained by cell-free protein synthesis in D₂O, as the serine hydroxymethyltransferase, that is naturally present in E. coli cell extracts, selectively replaces the pro-2S proton of glycine by a deuterium. To encourage the conversion by serine hydroxymethyltransferase, we performed the cell-free reaction without the addition of any glycine, exploiting the capability of the enzyme to convert serine to glycine with the help of tetrahydrofolate. ¹³C-HSQC spectra of ubiquitin produced with ¹³C/¹⁵N-serine showed that about a quarter of the glycine residues derived from serine were stereospecifically deuterated. Pulse sequences are presented that select the signals from the stereospecifically deuterated glycine residues.     

A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment

A new strategy of backbone resonance assignment is proposed based on a combination of the most sensitive TROSY-type triple resonance experiments such as TROSY-HNCA and TROSY-HNCO with a new 3D multiple-quantum HACACO experiment. The favourable relaxation properties of the multiple-quantum coherences and signal detection using the ¹³C antiphase coherences optimize the performance of the proposed experiment for application to larger proteins. In addition to the ¹H^N, ¹⁵N,¹³C and ¹³C chemical shifts the 3D multiple-quantum HACACO experiment provides assignment for the ¹H resonances in constrast to previously proposed experiments for large proteins. The strategy is demonstrated with the 44 kDa uniformly ¹⁵N,¹³C-labeled and fractionally 35% deuterated trimeric B. subtilis Chorismate Mutase measured at 20°C and 9°C. Measurements at the lower temperature indicate that the new strategy can be applied to even larger proteins with molecular weights up to 80 kDa.     

A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis

Sulfhydryl-containing proteins can oxidize during SDS-polyacrylamide gel electrophoresis to produce broad bands with irreproducible mobilities. Oxidation affects molecular weight estimates and decreases resolution. Alkylation of reduced proteins prior to electrophoresis eliminates the problems caused by reoxidation. A simple protocol involves reducing proteins with low concentrations of dithioerythritol and alkylating with a slight excess of iodoacetamide. These reactions are insensitive to atmospheric oxygen and to reagent concentrations provided that sulfhydryl groups are first completely reduced and then completely alkylated. The reagents need not be removed from the proteins prior to electrophoresis.     

Automatic methyl assignment in large proteins by the MAGIC algorithm

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics and function of biomolecules by NMR. Despite spectacular progress in the field, such studies remain rather limited in number. One of the main obstacles remains the assignment of the methyl resonances, which is labor intensive and error prone. Typically, NOESY crosspeak patterns are manually correlated to the available crystal structure or an in silico template model of the protein. Here, we propose methyl assignment by graphing inference construct, an exhaustive search algorithm with no peak network definition requirement. In order to overcome the combinatorial problem, the exhaustive search is performed locally, i.e. for a small number of methyls connected through-space according to experimental 3D methyl NOESY data. The local network approach drastically reduces the search space. Only the best local assignments are combined to provide the final output. Assignments that match the data with comparable scores are made available to the user for cross-validation by additional experiments such as methyl-amide NOEs. Several NMR datasets for proteins in the 25–50 kDa range were used during development and for performance evaluation against the manually assigned data. We show that the algorithm is robust, reliable and greatly speeds up the methyl assignment task.     

A simple method for the determination of reduction potentials in heme proteins

We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (E_m = −115 mV ± 2 mV, pH 7.0) and human CLOCK (E_m = −111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.     

A powerful method of sequential proton resonance assignment in proteins using relayed 15N-1H multiple quantum coherence spectroscopy

A powerful method of sequential resonance assignment of protein 1H-NMR spectra is presented and illustrated with respect to the DNA-binding protein ner from phage Mu. It is based on correlating proton-proton through-space and through-bond connectivities with the chemical shift of the directly bonded 15N atom. By this means, ambiguities arising from chemical shift degeneracy of amide proton resonances can be resolved. The experiments described involve combining the 1H-detected heteronuclear multiple quantum coherence correlation experiment with homonuclear nuclear Overhauser enhancement, J-correlated or Hartmann-Hahn experiments.     

MAP-XSII: an improved program for the automatic assignment of methyl resonances in large proteins

NMR studies of large proteins have gathered much interest in recent years, especially after methyl-transverse relaxation optimized spectroscopy was successfully applied to systems as large as ~1 MDa in molecular weight. However, to fully take advantage of these spectra, there is a need for convenient and robust methods for making resonance assignments rapidly. Here, we present an improved version of our program MAP-XS (methyl assignment prediction from X-ray structure) for the automatic assignment of methyl peaks, based on nuclear Overhauser effects (NOE) correlations and chemical shifts together with available structures. No manual analysis of the NOE data is needed in this new version, which helps to further accelerate the assignment process. A refined algorithm as well as more efficient sampling produces results from single runs of MAP-XSII using unanalyzed NOE data are comparable to those achieved by the old version using manually curated data with every NOE peak correctly attributed to the two related methyl peaks; in addition, checking the results from multiple parallel runs against each other provides an effective mechanism for getting rid of the wrong assignments while keeping the correct ones, which significantly improves the reliability of final assignments. The new program is tested against three different proteins and delivers ~95 % correct assignments; positive results are also achieved for tests using different cut-off distances for NOEs, structures of lower resolutions, and ambiguous residue types.     

A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides

The use of the ammonia deprotection step to introduce ¹⁵N labels at specific exocyclic amino positions of adenine, cytosine, guanine or 2-aminopurine of oligodeoxynucleotides is described.     

A simple method for discriminating between cell membrane and cytosolic proteins

* Transgenic plants expressing either green fluorescent protein (GFP)-genomic DNA or GFP-cDNA fusions have been used as powerful tools to define the subcellular localization of many proteins. Because most plant cells are highly vacuolated, the cytosol is confined to a thin layer at the periphery of the cells, making it very difficult to distinguish among cell wall, cell membrane and cytosolic GFP-fusion proteins. * Plasmolysis tests inform about cell-wall localization of GFP-tagged proteins, but they do not discriminate between its cell membrane and/or cytoplasmic localization. By observing the GFP signal in transgenic protoplasts placed at a hypotonic solution, it was possible to distinguish between cell membrane and cytosolic GFP-tagged proteins. * The osmotic disruption of the protoplast vacuole in the hypotonic solution allows the diffusion of the GFP signal from the cell periphery to the central part of the cell volume when the GFP is fused to a soluble protein. By contrast, such diffusion does not occur when the protein under study is attached to the cell membrane. * The present method is easier, faster and cheaper than subcellular fractionating studies and/or immunoelectron microscopy, which have been traditionally used to discern between cell membrane and cytosolic proteins.     

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of ¹³C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of ¹³C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-¹³C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally ¹³C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.     

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.     

A simple method for the separation of bile acid groups by ion-exchange chromatography

A simple ion-exchange chromatographic method for the separation of bile acid mixtures is described. The method employs Dowex 1 as anion exchanger and mixtures of aqueous ethanol and hydrochloric acid as eluants. The use of mixtures in which both the ethanol and acid concentrations are varied has permitted a clear separation of the major bile acid groups (nonconjugated, glycine conjugated, and taurine conjugated bile acids).     

A simple way for sequential assignment in isotopically enriched proteins using a H(N)CACO correlation

Summary A simplified scheme for sequential assignment in isotopically enriched proteins is presented. It is based on the standard triple resonance experiments HNCO, HN(CO)CA, HNCA and a modified H(N)CACO correlation, in which both of the H^N connectivities to the CA/C pair of residue i and i-1 are observed. The H(N)CACO was tested on uniformly ¹³C/¹⁵N enriched P13 domain of mannose permease (31 kDa).     

HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins

A TROSY-based triple-resonance pulse scheme is described which correlates backbone ¹H and ¹⁵N chemical shifts of an amino acid residue with the ¹⁵N chemical shifts of both the sequentially preceding and following residues. The sequence employs ¹ J _NC and ² J _NC couplings in two sequential magnetization transfer steps in an `out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly ²H/¹³C/¹⁵N-labelled proteins in the 30-kDa range.     

A simple in vitro method for raising monoclonal antibodies to cochlear proteins.

Monoclonal antibodies have been produced to mammalian hair cell antigens using a simple in vitro kit. Antigen was crudely prepared from dissected cochlear tissue by detergent extraction. There was no need to purify hair cells. Hybridoma supernatents were screened most efficiently on dissociated cells fixed with acetone. The immunisation method is sensitive to nanograms of antigen and can generate responses to conserved or weak antigens. The kit requires very little previous experience with cell culture and generates monoclonal antibodies within 3-4 weeks. It has overcome a number of problems with production of antibodies to hair cells and it should prove to be a very valuable tool in many laboratories.