Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.     

13C nuclear magnetic resonance studies on selectively labeled bacterial biofilms

Bacterial biofilms of Pseudomonas aeruginosa selectively labeled by introduction of 2-¹³C-glycerol was studied by solid-state and high-resolution nuclear magnetic resonance. The ¹³C nuclei were mainly integrated into mannuronate and guluronate, the two monomer units forming the bacterial alginate. The signal for the C5 position of the mannuronate, which was easily identified and well separated from other peaks, was analyzed for molecular mobility. The result indicated a high degree of motional freedom within the molecular network of the alginate. Despite the fact that the alginate was part of a solid aqueous gel phase, the reorientation mechanism of the monomer units came close to isotropic tumbling. Solid-state spectra of biofilms labeled in the described manner may serve as a valuable tool for noninvasive analyses of molecular mobility of the alginate component under various influences, thereby revealing important structural information. In addition, the effect of a monovalent electrolyte (LiCl) on the molecular mobility of alginate fragments in an aqueous solution was studied by determining the spin–lattice relaxation times, line widths and line shapes under variations of the ion concentration. The presence of ions accelerated overall motions but left rapid local motions virtually unaffected. Journal of Industrial Microbiology & Biotechnology (2001) 26, 62–69. Received 26 January 2000/ Accepted in revised form 30 August 2000     

NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands

Selective isotopic labeling of larger proteins greatly simplifies protein NMR spectra and reduces signal overlap, but selectively labeled proteins cannot be easily assigned since the sequential assignment method is not applicable. Here we describe a strategy for resonance assignment in selectively labeled proteins. Our approach involves a spin-labeled analog of a ligand of which the three-dimensional structure in complex with the target protein is known. Other methods for introduction of the spin label are possible. The paramagnetic center causes faster relaxation of all neighboring nuclei in a distance-dependent manner. Measurement of this effect allows to deduce distances between isotopically labeled residues and the paramagnetic center which can be used for resonance assignment. The method is demonstrated for the catalytic domain of Abl kinase in complex with the inhibitor, STI571.     

Refolding of ribonuclease A monitored by real-time photo-CIDNP NMR spectroscopy

Photo-CIDNP NMR spectroscopy is a powerful method for investigating the solvent accessibility of histidine, tyrosine and tryptophan residues in a protein. When coupled to real-time NMR, this technique allows changes in the environments of these residues to be used as a probe of protein folding. In this paper we describe experiments performed to monitor the refolding of ribonuclease A following dilution from a high concentration of chemical denaturant. These experiments provide a good example of the utility of this technique which provides information that is difficult to obtain by other biophysical methods. Real-time photo-CIDNP measurements yield residue-specific kinetic data pertaining to the folding reaction, interpreted in terms of current knowledge of the folding of bovine pancreatic ribonuclease A.     

High-yield expression of isotopically labeled peptides for use in NMR studies

Fusion protein constructs of the 56 amino acid globular protein GB-1 with various peptide sequences, coupled with the incorporation of a histidine tag for affinity purification, have generated high-yield fusion protein constructs. Methionine residues were inserted into the constructs to generate pure peptides following CNBr cleavage, yielding a system that is efficient and cost effective for isotopic labeling of peptides for NMR studies and other disciplines such as mass spectroscopy. Six peptides of varying sequences and hydrophobicities were expressed using this GB-1 fusion protein technique and produced soluble fusion protein constructs in all cases. The ability to easily express and purify recombinant peptides in high yields is applicable for biomedical research and has medicinal and pharmaceutical applications.     

Sequential assignment of 1H, 13C and 15N resonances of human carbonic anhydrase I by triple-resonance NMR techniques and extensive amino acid-specific 15N-labeling

Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific ¹⁵N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A.     

Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans

The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30–40 mg/l in LB, 15–20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with ¹⁵N, ¹⁵N/¹³C and even ¹⁵N/¹³C/²H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.     

High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies

The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure.     

A rapid method to attain isotope labeled small soluble peptides for NMR studies

A widely applicable strategy is presented for efficient and rapid production of small water soluble peptides expressed as fusion proteins with the immunoglobulin-binding domain of streptococcal protein G. A simple extraction and purification scheme that includes a protease cleavage step to release the target peptide is described. The yield of authentic target peptide exceeds 10 mg per liter of culture. Production of U-¹³C, ¹⁵N and highly deuterated U-¹³C, ¹⁵N isotope labeled peptide is demonstrated for the 11 residue S2 peptide, corresponding to the C-terminus of the -subunit of transducin, and the coiled coil trimerization domain from cartilage matrix protein (CMPcc), respectively. Heteronuclear two-dimensional NMR spectra are used for initial peptide characterization.     

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

Acid-induced denaturation of Escherichia coli ribonuclease HI analyzed by CD and NMR spectroscopies

Acid-induced denaturation of the ribonuclease HI protein from Escherichia coli was analyzed by CD and NMR spectroscopies. The CD measurement revealed that the acid denaturation at 10 degrees C proceeds from the native state (N-state) to a molten globule-like state (A-state), through an apparently more unfolded state (U(A)-state). In (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, cross peaks from the N-state and those from the other two states are distinctively observed, while the U(A)-state and A-state are not distinguished from each other. Cross peaks from the U(A)/A-states showed a small pH dependence, which suggests a similarity in the backbone structure between the two states. The direct hydrogen-deuterium (H-D) exchange measurement at pH with the largest population of U(A)-state revealed that at least alpha-helix I is highly protected in the structure of the U(A)-state. A pH-jump H-D exchange analysis showed that the protection of alpha-helix I is highest also in the A-state. The profile of hydrogen-bond protection indicated that the structure of the A-state is closely related to that of the kinetic folding intermediate.     

A One-Dimensional (Proton and Phosphorus) and Two-Dimensional (Proton) In Vivo NMR Spectroscopic Study of Reversible Global Cerebral Ischemia

Abstract: The suitability of two-dimensional (2D) proton spectroscopy for monitoring, in vivo, the changes in levels of brain metabolites induced by cerebral ischemia was investigated in an experimental model of 30-min reversible ischemia induced by four-vessel occlusion in the rat. The resulting data were compared with those obtained by one-dimensional (1D) proton and phosphorus spectroscopy. Phosphorus spectra obtained during ischemia showed significant drops in levels of phosphocreatine (−73%), β-ATP (−60%), and intracellular pH (to 6.30) and an increase in inorganic phosphate level (905%). 1D and 2D proton spectra showed decreases in the N -acetylaspartate/creatine-phosphocreatine ratio that were not significantly different [−21% (1D) and −32% (2D)]. Similarly, the increases in lactate/creatine-phosphocreatine ratio were not significantly different [2,546% (1D) and 3,020% (2D)]. 2D spectroscopy also indicated a decrease in aspartate (−66%) and an increase in the inositol-choline derivative (+124%) pools during ischemia and an increase in alanine pool (+516%) during reperfusion. The glutamate-glutamine pool and taurine content did not change significantly during ischemia but decreased during reperfusion. The glucose level transiently decreased (−67%) during ischemia and increased immediately after (+261%). The levels of all the metabolites investigated returned to control values within 175 min after ischemia. 2D spectroscopy seems to be a reliable method of monitoring the changes in levels of cerebral compounds known to be involved in ischemia.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies

Human macrophage elastase (MMP-12) plays an important role in inflammatory processes and is involved in a number of physiological or pathological situations, such as conversion of plasminogen into angiostatin, allergic airway inflammation, vascular remodeling or alteration, as well as emphysema, and has been justified as a novel drug target. Here, we report the over-expression in Escherichia coil, purification and refolding of MMP-12 catalytic domain for NMR studies. The primary sequence of expressed protein was identified by means of MALDI-TOF MS, and was confirmed by the MALDI-TOF MS data of trypsin-digested peptides. A significantly optimized protocol has been worked out to prepare 15N and/or 13C-labeled MMP-12 catalytic domain, and the yield of the purified protein is estimated to 10-12 mg from 0.5L of M9 minimal media. Finally, the 15N-1H HSQC spectrum of uniformly 15N-labeled MMP-12 catalytic domain indicates the presence of well-ordered and properly folded protein in a monomeric form.     

A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells

Whereas bacterial expression systems are widely used for production of uniformly or selectively ¹⁵N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively ¹⁵N-labeled proteins in insect cells. The quantities of ¹⁵N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the ¹⁵N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.     

Selective `unlabeling' of amino acids in fractionally 13C labeled proteins: An approach for stereospecific NMR assignments of CH3 groups in Val and Leu residues

A novel methodology for stereospecific NMR assignments of methyl (CH₃) groups of Val and Leu residues in fractionally ¹³C-labeled proteins is presented. The approach is based on selective `unlabeling' of specific amino acids in proteins while fractionally <sup>13</sup>C-labeling the rest. A 2D [<sup>13</sup>C-<sup>1</sup>H] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the `unlabeled' amino acid residues. Such spectral simplification aids in unambiguous stereospecific assignment of diastereotopic CH₃ groups in Val and Leu residues in large proteins. This methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP).     

An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins

Summary A biosynthetic strategy has recently been developed for the production of ¹⁵N, ¹³C, ²H-labeled proteins using ¹H₃C-pyruvate as the sole carbon source and D₂O as the solvent. The methyl groups of Ala, Val, Leu and Ile (2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a ¹⁵N, ¹³C, ²H, ¹H₃C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range.