Electron paramagnetic resonance spectroscopy of thyroid peroxidase

Thyroid peroxidase was isolated from porcine thyroids by two methods. Limited trypsin proteolysis was employed to obtain a cleaved enzyme, and affinity chromatography was used to isolate intact thyroid peroxidase. Enzyme isolated by both methods was used in the examination of the heme site of native thyroid peroxidase and its complexes by EPR spectroscopy. Intact thyroid peroxidase showed a homogeneous high-spin EPR signal with axial symmetry, in contrast to the rhombic EPR signal of native lactoperoxidase. Reaction of cyanide or azide ion with native thyroid peroxidase resulted in the loss of the axial EPR signal within several hours. The EPR spectroscopy of the nitrosyl adduct of ferrous thyroid peroxidase exhibited a three-line hyperfine splitting pattern and indicated that the heme-ligand structure of thyroid peroxidase is significantly different from that of lactoperoxidase.     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.     

Ruthenium red as a paramagnetic probe in NMR spectroscopy

It is shown that ruthenium red acts as a paramagnetic probe in NMR spectroscopy. Unlike lanthanide and calcium ions it acts as a substitution probe for polyamine binding sites in biological systems, although it also binds at sites where calcium binds.     

Carbon-13 nuclear magnetic resonance spectroscopy of lipids: differential line broadening due to cross-correlation effects as a probe of membrane structure

We have obtained proton-coupled carbon-13 nuclear magnetic resonance (NMR) spectra of a variety of lipid-water and lipid-drug-water systems, at 11.7 T, as a function of temperature, using the "magic-angle" sample-spinning (MAS) NMR technique. The resulting spectra show a wide range of line shapes, due to interferences between dipole-dipole and dipole-chemical shielding anisotropy interactions. The differential line-broadening effects observed are particularly large for aromatic and olefinic (sp2) carbon atom sites. Coupled spectra of the tricyclic antidepressants desipramine and imipramine, in 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophases, show well-resolved doublets having different line shapes for each of the four aromatic methine groups, due to selective averaging of the four C-H dipolar interactions due to rapid motion about the director (or drug C2) axis. 2H NMR spectra of [2,4,6,8-2H4]desipramine (and imipramine) in the same 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophase exhibit quadrupole splittings of approximately 0-2 and approximately 20 kHz, indicating an approximate magic-angle orientation of the C2-2H(1H) and C8-2H(1H) vectors with respect to an axis of motional averaging, in accord with the 13C NMR results. Selective deuteration of imipramine confirms these ideas. Spectra of digalactosyl diglyceride [primarily 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl ]-3- (alpha-D-galactopyranosyl-1-6-beta-D-galactopyranosyl)-sn-glycerol]-H2O (in the L alpha phase) show a large differential line broadening for C9 but a reduced effect for C10, consistent with the results of 2H NMR of specifically 2H-labeled phospholipids [Seelig, J., & Waespe-Saracevic, N. (1978) Biochemistry 17, 3310-3315].(ABSTRACT TRUNCATED AT 250 WORDS)     

Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance, and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase.

The interactions of gadolinium ion, lithium, and two substrate analogues, beta,gamma-imido-ATP (AMP-PNP) and tridentate CrATP, with the calcium ion transport adenosine triphosphatase (Ca2+-ATPase) of rabbit muscle sarcoplasmic reticulum have been examined by using 7Li+ NMR, water proton NMR, and Gd3+ EPR studies. Steady-state phosphorylation studies indicate that Gd3+ binds to the Ca2+ activator sites on the enzyme with an affinity which is approximately 10 times greater than that of Ca2+. 7Li+, which activates the Ca2+-ATPase in place of K+, has been found to be a suitable nucleus for probing the active sites of monovalent cation-requiring enzymes. 7Li+ nuclear relaxation studies demonstrate that the binding of Gd3+ ion to the two Ca2+ sites on Ca2+-ATPase increases the longitudinal relaxation rate (1/T1) of enzyme-bound Li+. The increase in 1/T1 was not observed in the absence of enzyme, indicating that the ATPase enhances the parmagnetic effect of Gd3+ on 1/T1 of 7Li+. Water proton relaxation studies also show that the ATPase binds Gd3+ at two tight-binding sites. Titrations of Gd3+ solutions with Ca2+-ATPase indicate that the tighter of the two Gd3+-binding sites (site 1) provides a ghigher enhancement of water relaxation than the other, weaker Gd3+ site (site 2) and also indicate that the average of the enhancements at the two sites is 7.4. These data, together with a titration of the ATPase with Gd3+ ion, yield enhancements, epsilonB, of 9.4 at site 1 and 5.4 at site 2. Analysis of the frequency dependence of 1/T1 of water indicates that the electron spin relaxation taus of Gd3+ is unusually long (2 X 10(-9) s) and suggests that the Ca2+-binding sites on the ATPase experience a reduced accessiblity of solvent water. This may indicate that the Ca2+ sites on the Ca2+-ATPase are buried or occluded within a cleft or channel in the enzyme. The analysis of the frequency dependence is also consistent with three exchangeable water protons on Gd3+ at site 1 and two fast exchanging water protons at site 2. Addition of the nonhydrolyzing substrate analogues, AMP-PNP and tridenate CrATP, to the enzyme-Gd3+ complex results in a decrease in the observed enhancement, with little change in the dipolar correlation time for Gd3+, consistent with a substrate-induced decrease in the number of fast-exchanging water protons on enzyme-bound Gd3+. From the effect of Gd3+ on 1/T1 of enzyme-bound Li+, Gd3+-Li+ separations of 7.0 and 9.1 A are calculated. On the assumption of a single Li+ site on the enzyme, these distances set an upper limit on the separation between Ca2+ sites on the enzyme of 16.1 A.     

Characterization of metabolic profile of intact non-tumor and tumor breast cells by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy

¹H high-resolution magic angle spinning nuclear magnetic resonance (¹H HR–MAS NMR) spectroscopy was used to analyze the metabolic profile of an intact non-tumor breast cell line (MCF-10A) and intact breast tumor cell lines (MCF-7 and MDA-MB-231). In the spectra of MCF-10A cells, six metabolites were assigned, with glucose and ethanol in higher concentrations. Fifteen metabolites were assigned in MCF-7 and MDA-MB-231 ¹H HR–MAS NMR spectra. They did not show glucose and ethanol, and the major component in both tumor cells was phosphocholine (higher in MDA-MB-231 than in MCF-7), which can be considered as a tumor biomarker of breast cancer malignant transformation. These tumor cells also show acetone signal that was higher in MDA-MB-231 cells than in MCF-7 cells. The high acetone level may be an indication of high demand for energy in MDA-MB-231 to maintain cell proliferation. The higher acetone and phosphocholine levels in MDA-MB-231 cells indicate the higher malignance of the cell line. Therefore, HR–MAS is a rapid reproducible method to study the metabolic profile of intact breast cells, with minimal sample preparation and contamination, which are critical in the analyses of slow-growth cells.     

Photo-CIDNP nuclear magnetic resonance as a probe for conformational changes in epidermal growth factor

Photo-chemically induced dynamic nuclear polarization (CIDNP)-NMR spectroscopy at 360 MHz has been used to investigate pH-induced conformational transitions in mouse epidermal growth factor. At about pH 9, all five tyrosine residues and both tryptophan residues are, to various extents, solvent-exposed, while the His-22 residue is buried in the protein matrix. Tyr-13 is the least exposed of the tyrosine residues and also the most immobilized. As the pH is decreased to 5.9, the tryptophan residues gradually become less exposed, while the Tyr-13 residue becomes internalized in the protein. These data suggest that the C-terminus and part of the N-terminal structural domain are affected by a conformational transition in mouse epidermal growth factor occurring between pH 6 and 8 via breakage of the His-22 inter-residue linkage. Above pH 9, a decreased photo-CIDNP effect is evident for both tryptophans and for Tyr-10 and Try-13; this information suggests that a second conformational change takes place at basic pH, which may simply be incipient denaturation.     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein-ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein-ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein-ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein-ligand interactions in vivo.     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein–ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein–ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein–ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein–ligand interactions in vivo.     

Electron paramagnetic resonance spectroscopy as a monitor of the pathway of the thermal unfolding of ribonuclease A.

α-L-Fucosidase activity is elevated in skin fibroblasts from cystic fibrosis patients when compared to controls. The activities of nine other acid hydrolases including neuraminidase are similar in cystic fibrosis and control fibroblasts. The relationship of these results to the recent finding of a decreased activity of α-L-fucosidase in the serum of cystic fibrosis patients is discussed. It is proposed that an abnormal distribution of α-L-fucosidase is involved in the pathogenesis of this disease.     

Low temperature magnetic circular dichroism spectroscopy as a probe for the optical transitions of paramagnetic nickel in hydrogenase

A partially-purified sample of hydrogenase from Methanobacterium thermoautotrophicum (delta H strain) has been investigated by optical absorption, magnetic circular dichroism and electron paramagnetic resonance spectroscopy. Variable temperature magnetic circular dichroism studies reveal, for the first time, the optical transitions associated with the Ni(III) center in the oxidized enzyme. Low temperature magnetic circular dichroism spectroscopy provides a new method of assessing both the coordination environment of Ni in hydrogenase and the appropriateness of inorganic model complexes.     

19F nuclear magnetic resonance as a probe of structural transitions and cooperative interactions in heavy meromyosin

An 19F NMR probe has been attached to the reactive sulfhydryl SH1 of the globular heads of rabbit skeletal heavy meromyosin. It serves as a sensitive monitor of the conformational state of the heads of heavy meromyosin in a manner similar to that seen for subfragment-1 (Shriver, J.W., and Sykes, B.D. (1982) Biochemistry 21, 3022-3028; Tollemar, U., Cunningham, K., and Shriver, J.W. (1986) Biochim. Biophys. Acta 873, 243-251). The NMR spectra indicate that there are at least two states for the heads in the SH1 region. The energetics of the interconversion of the two states of heavy meromyosin (HMM) differs significantly from that of S-1. In HMM in the absence of divalent cations, there are two reversible paths between the low temperature and high temperature states with a hysteresis-like behavior. One path is consistent with the head groups behaving independently and similar to S-1 alone. The second path indicates a coupling of the globular head region observed in S-1 with a second region forming a distinctly different cooperative unit. Upon addition of Ca(II) the hysteresis effect is lost and only the second cooperative unit is observed. Two explanations are offered for these results: the globular heads in HMM may couple with the S-2 segment, or the two globular heads of HMM may couple to form a larger cooperative unit. The ability to stabilize the larger cooperative unit with a divalent metal ion implicates a role for the LC2 light chain in coupling regions of the myosin molecule.     

Rubredoxin as a paramagnetic relaxation-inducing probe

The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c₃ was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2¹ and M18¹. The rubredoxin binding surface in the complex with cytochrome c₃ was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c₃ are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in ¹HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes.     

Electron paramagnetic resonance spectroscopy on G-protein-coupled receptors: Adopting strategies from related model systems

Membrane proteins, including ion channels, transporters and G-protein-coupled receptors (GPCRs), play a significant role in various physiological processes. Many of these proteins are difficult to express in large quantities, imposing crucial experimental restrictions. Nevertheless, there is now a wide variety of studies available utilizing electron paramagnetic resonance (EPR) spectroscopic techniques that expand experimental accessibility by using relatively small quantities of protein. Here, we give an overview starting from basic strategies in EPR on membrane proteins with a focus on GPCRs, while emphasizing several applications from recent years. We highlight how the arsenal of EPR-based techniques may provide significant further contributions to understanding the complex molecular machinery and energetic phenomena responsible for seamless workflow in essential biological processes.     

Phosphorus-31 nuclear magnetic resonance spectroscopy of human retinoblastoma cells: correlations with metabolic indices

The ³¹P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by ³¹P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1,2 and 3 days post feeding showed a 40% decrese in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.