Lawrence J Berliner

• University of Denver

Since the first EPR/ESR spectrum of a paramagnetic substance was published over 70 years ago, the technical improvements did not occur until after/during World War II with the advent of radar technology. The approaches to biomedical problems started somewhat later with the real burst of activity starting after the birth of the spin label technique...

Environmental chemistry and toxicology was the topic of two different, yet similar style courses: one an organized, one-term on-campus course and the other a two-week intensive travel course in Thailand. The format was always the flipped classroom, cooperative learning model, where prechosen student groups prepared oral presentations on subjects fr...

Introduction Tabulations Concluding Remarks References

Singlet molecular oxygen produced in the presence of 2,2,6,6-tetramethyl-4-piperidone (TMPD) in 1,4-dioxan, by boron subphthalocyanine chloride as the sensitizer, was monitored by Electron Paramagnetic Resonance (EPR) via the oxidation of TMPD to the corresponding aminoxyl radical. This radical product is formed in low yields along the singlet oxyg...

IntroductionThe early yearsAdvantages of nitroxidesApplications of spin labeling to biochemical and biological systemsDistance measurementsSite directed spin labeling (SDSL): how is it done?Other spin labeling applicationsConclusions References

The physical principles that underlie organic reactions were established by a systematic study of chemical reaction dynamics that employed correlated measurements of reaction rates and a physical parameter that could be related of the electronic properties of the molecules in question (Hammett, 1970). Today, molecular science emphasizes the concept...

The spin-lattice, or longitudinal, relaxation time T 1 plays an important role in magnetic resonance because it provides significant information about the coupling of a paramagnetic ion with its environment via its dependence on such factors as temperature, frequency (Scott & Jefferies, 1962; Kurtz & Stapleton, 1980), spin concentration (Gill, 1962...

The measurement of very short spin-lattice, or longitudinal, relaxation (SLR) times (i.e., 10−10 < T 1 < 10−6 s) is of great importance today for the study of relaxation processes. Recent case studies include, for example, glasses doped with paramagnetic ions (Vergnoux et al., 1996; Zinsou et al., 1996), amorphous Si (dangling bonds) and copper-chr...

Our current knowledge about biological membranes shows that they belong to the most important cell structures. Mass transport and signal transduction obviously appear to be vital to physiological functions of biomembranes as they enable cellular compartmentalization and control over it at the same time. Many experiments and theoretical consideratio...

Instrumentation aspects of ESR spectroscopy are treated thoroughly in a standard text (Poole 1997). However, a majority of ESR users are nowadays concerned with application work rather than development of instrumentation and methods. For such work, a comprehensive knowledge of instrumentation aspects is not required, yet a basic knowledge is needed...

The main emphasis of this chapter, as well as of the whole volume, is on sitedirected spin-labeling (SDSL) and spin-labeling ESR as an alternative and a complementary structural technique for NMR and x-ray diffraction of proteins and other complex biomolecular systems. The spin labeling method is particularly powerful for elucidating local protein...

ESR Spectroscopy in Membrane Biophysics Dr. Marcus A. Hemminga, Wageningen University Dr. Lawrence J. Berliner, University of Denver Membrane proteins offer the greatest challenge in structural biology, and there is an urgent need to develop and apply new biophysical methodologies that are able to generate detailed structural information. Among mod...

It was recently shown that alpha-lactalbumin interacts with histones and simple models of histone proteins such as positively charged polyamino acids, suggesting that some fundamental aspects of the protein surface electrostatics may come into play. In the present work, the energies of charge-charge interaction in apo- and Ca(2+)-loaded alpha-lacta...

It was recently shown that alpha-lactalbumin associated with oleic acid (HAMLET) interacts with core histones thereby triggering apoptosis of tumor cells (J. Biol. Chem. 2003, 278, 42131). In previous work, we revealed that monomeric alpha-lactalbumin in the absence of fatty acids can also interact with histones and, moreover, with basic poly-amino...

The study of small Asp-Phe analogs was undertaken since this dipeptide sequence is critical in fibrinogen recognition and catalysis. The inhibition of clotting activity by Asp-Phe-methyl ester (aspartame), formyl-Asp-Phe-methyl ester and acetyl-Asp-Phe was biphasic in all cases, indicating the presence of at least two binding sites. The N-terminall...

The applications of in vivo electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy have been impressive over a relatively short period despite the many obstacles which had to be overcome, such as dielectric absorption and biodestruction. The loop-gap resonators and modified loop coil systems have emerged as the most sui...

The measurement of nitric oxide (NO) in biological samples has normally required destructive chemical techniques. The ability to detect NO non-invasively in living animals or excised organs has great potential using specialized electron paramagnetic resonance (EPR) methods. Although NO is paramagnetic, it cannot be observed directly unless it is co...

Experiments using purified recombinant human NAD(P)H:quinone oxidoreductase 1 (NQO1) revealed that the auto-oxidation of fully reduced protein resulted in a 1:1 stoichiometry of oxygen consumption to NADH oxidation with the production of hydrogen peroxide. The rate of auto-oxidation of fully reduced NQO1 was markedly accelerated in the presence of...

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl rad...

Alpha-lactalbumin (alpha-LA) is biosynthesized and stored at the smooth endoplasmic reticulum (ER), then transferred to the Golgi lumen when prolactin stimulation of lactose biosynthesis and secretion takes place. Because both environments are composed of membranes, it was of interest to examine the interactions of alpha-LA with relevant model and...

Electron magnetic resonance spectroscopy is undergoing something akin to a renaissance that is attributable to advances in microwave circuitry and signal processing software. EPR: Instrumental Methods is a textbook that brings the reader up to date on these advances and their role in providing better experimental techniques for biological magnetic...

The aim of this chapter is to review the various methods used to detect NO and an in-depth overview of in vivo and in vitro EPR methods.

The role of nitric oxide (NO) in epileptogenesis was studied in pentylenetetrazole (PTZ)-treated animals using in vivo and ex vivo EPR spectroscopy. NO generation was measured directly in the brain of a PTZ-induced mouse in vivo by an L-band EPR spectrometer. An elevation in NO production in the brain was observed during convulsions, and more NO wa...

The enzymatic generation of nitric oxide (NO) in vivo has been reported to be modulated by ions, such as copper and iron. Superparamagnetic iron oxide (SPIO) or ferumoxides is a liver-specific magnetic resonance contrast agent that is taken up by the Kupffer cells, where NO is generated by inducible nitric oxide synthase (iNOS). Thus, it is importa...

The ultimate goal of in vivo electron spin resonance (ESR) spin trapping is to provide a window to the characterization and quantification of free radicals with time within living organisms. However, the practical application of in vivo ESR to systems involving reactive oxygen radicals has proven challenging. Some of these limitations relate to ins...

α-Lactalbumin (α-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human α-LA, but zinc binding had never been measured in solution for human α-LA. This residue w...

Small milk protein α-lactalbumin (α-LA), a component of lactose synthase, is a simple model Ca2+ binding protein, which does not belong to the EF-hand proteins, and a classical example of molten globule state. It has a strong Ca2+ binding site, which binds Mg2+, Mn2+, Na+, and K+, and several distinct Zn2+ binding sites. The binding of cations to t...

The effects of amino acid substitutions in the N-terminus of bovine recombinant α-lactalbumin (including enzymatic removal of the N-terminal methionine and deletion of Glu-1) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC). Wild-type recombinant α-lactalbumin has a lower thermostabil...

Native β-lactoglobulin (Blg) binds 1 mole of palmitic acid per mole of protein with a dissociation constant of 0.6 μM for the primary fatty acid binding site. Chemical modification of Cys 121, which lies at the external putative hydrophobic binding site of Blg, does not affect retinol or 4,4′-bis 1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) bin...

Bovine α-lactalbumin (α-LA) has been shown by intrinsic protein fluorescence and electron spin resonance methods to interact with the spin-labeled fatty acid analog, 5-doxylstearic acid, as well as stearic acid. An intrinsic fluorescence titration of various α-LA forms with 5-doxylstearic acid causes first an increase and then a decrease in emissio...

α-Lactalbumin possesses multiple Zn2+ binding sites, with the strongest site having an affinity constant of 5×105 M−1 [Permyakovet al. (1991),J. Protein Chem. 100, 577]. The binding of zinc at secondary sites is accompanied by destabilization of the protein structure and progressive protein aggregation. This pronounced destabilization is reflected...

Tissue plasminogen activator (t-PA) is an exceptional serine protease, because unlike most other serine protease zymogens single-chain tissue plasminogen activator (sct-PA) possesses a substantial amount of proteolytic activity. The unusual reaction of sct-PA afforded the opportunity to directly compare the active site environment of sct-PA and two...

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5C decrease in the following order: Ca(II)--LA>Zn(II), Ca...

The strong calcium-binding site of α-lactalbumin comprises the carboxylate side chains of aspartic acid 82, 87, and 88 and the carbonyl oxygens of residues 79 and 84. A single methionine residue was selectively modified by controlled CNBr cleavage to yield homoserine at position 90. The CNBr-cleaved α-lactalbumin lost the ability to bind calcium st...

EPR imaging was applied to the study of several important processes in polymers and materials. The solvent diffusion and induced swelling was visualized in the water/DMF or toluene/DMF interactions with polycarbonate or polystyrene molded rods. The cross-sectional images revealed non-homogeneous solute/solvent diffusion, rod expansion and crack dev...

Diffusion processes in biological tissue are important problems for noninvasive investigation. As a model study, this work addresses the diffusion of an electrolyte buffer (Krebs) solution containing a nitroxide spin probe into a cylindrical polyacrylamide gel rod. The nitroxide spin density distribution was imaged at 1.6 GHz in gel cross sections...

One of the most important challenges in the field of structural biology and structural proteomics is the study of membrane proteins. These proteins reside in cell and organelle membranes and govern many important cellular functions, including cell signaling, membrane potential stabilization, energy transduction, pH and cell volume regulation, all o...

ESR spectroscopy of site-directed spin-labeled biomolecules (Site-Directed Spin Labeling, SDSL) has emerged as a powerful method for studying the structure and conformational dynamics of proteins and nucleic acids under conditions relevant to function (for reviews see, e.g., Feix and Klug 1998; Hubbell et al. 1996; Hubbell et al. 1998, 2002). In th...

The recent resurgence of ESR (electron spin resonance) in structural biology is in large part due to the development of distance measurements. This application was made possible by targeting of specific sites/domains by cysteine mutagenesis. Four techniques were developed to cover various distance ranges: exchange ESR for the very short distances (...