Infrared spectroscopy of proteins.

I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

1. Electron Donor 2223 2. Electron Transfer Path 2225 3. Electron Transfer Mechanism 2226 4. Physiological Relevance 2226 D. Radical Reactions in Photolyase 2226 E. “Dark Function” of Photolyase 2227 F. Regulation of Photolyase 2227 III. (6−4) Photolyase 2228 IV. Cryptochromes 2230 A. Structure 2231 B. Function 2231 1. Circadian Rythm 2231 2. Mammalian Circadian System 2231 3. Cryptochromes and the Circadian Clock 2232 V. Perspectives 2234 VI. Acknowledgment 2235 VII. References 2235

Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors.

https://www.cell.com/developmental-cell/pdf/S1534-5807(05)00421-1.pdf

Hyperdynamic Plasticity of Chromatin Proteins in Pluripotent Embryonic Stem Cells

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation Here we show that HP1α, -β, and -γ are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase These findings establish a regulatory mechanism of protein–protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark

Regulation of HP1–chromatin binding by histone H3 methylation and phosphorylation

Deazaflavins have been found to act as potent catalysts in the photoreduction of flavoproteins in the presence of EDTA and other "photosubstrates". In distinction to the catalysis brought about by normal flavins which involves dark reaction of the photoreduced flavin catalyst, the mechanism of the catalysis by deazaflavins has been shown to involve unstable, strongly reducing radicals which are generated by photolysis of a preformed covalent dimer. By this new method it is possible to reduce not only flavoproteins but a variety of other redox proteins, including heme proteins and iron-sulfur proteins. By virtue of its great catalytic efficiency, it is possible to employ concentrations of deazaflavin sufficiently low as not to interfere with the spectral evaluation of the reduced proteins obtained.

Photoreduction of flavoproteins and other biological compounds catalyzed by deazaflavins.

Flavins are very versatile coenzymes, functioning with considerable efficiency in a wide variety of enzymic reactions involving either two-electron or one-electron transfers, with both functions often catalysed by the same enzyme. They are responsible for catalysing the dehydrogenation of many different types of compounds, including dithiols, reduced nicotinamide nuclwtides, alcohols and a-hydroxy acids, amines and a-amino acids, and even saturated C-C bonds, provided that a suitable activating group such as a carbonyl residue is situated a/% to the bond to be oxidized. In the process of catalysing these dehydrogenation reactions, the flavin is itself reduced, and, in order to function catalytically, the oxidized form must be regenerated at the expense of reduction of some acceptor. The acceptor may be in some cases the oxidized form of the same type of compound that serves as reducing substrate, e.g. it might be a disulphide, an oxidized nicotinamide nucleotide or an unsaturated compound such as fumarate or crotonylCoA. In such a case the enzyme might conveniently be classified as a transhydrogenase and subclassified as a C-C, C-S, C-N or N-N transhydrogenase, depending on the nature of the atoms acting as hydrogen donor and hydrogen acceptor (Hemmerich & Massey, 1979). In most cases, however, the acceptor molecule will be molecular 0, or another redox protein, such as an iron-sulphur protein or a cytochrome. In the latter cases the flavoprotein necessarily acts as a mediator between two-electron and one-electron transfers. Flavoproteins fill a unique spot in biochemistry with this capacity. An equal richness of possibilities exists in the reactions of different flavoproteins with molecular 0,. In some cases one-electron transfer is carried out, with superoxide (02-) and flavin semiquinone as the immediate products of the reaction. In other cases a direct two-electron reduction of 0, to H,O, appears to occur, and in another class of flavoproteins, the mono-oxygenases, one atom of the 0, molecule is incorporated into H20 and the other is incorporated into another substrate of the enzyme, to form an oxygenated product.

Active-site probes of flavoproteins

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin Here we investigated the diffusional behaviors of HP1 isoforms in mammalian cells Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50-80%) are highly mobile (recovery halftime: t(1/2) approximately 09 s; diffusion coefficient: D approximately 06-07 microm(2) s(-1)) Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t(1/2) approximately 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5-7% of very slow HP1 molecules The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells During mitosis 8-14% of GFP-HP1alpha, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1alpha in heterochromatin of mitotic chromosomes These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle

High- and Low-mobility Populations of HP1 in Heterochromatin of Mammalian Cells

It has been found that small amounts of free flavins greatly accelerate the photochemical reduction of flavoproteins both to the radical and fully reduced oxidation states. This catalytic effect has been shown to be due to the rapid photochemical reduction of the free flavin to its fully reduced state, followed by its reaction with the flavoprotein to yield flavoprotein radical and by its reaction with flavoprotein radical to yield fully reduced flavoprotein. Evidence is presented that the same route may occur with flavoproteins in the absence of added flavins. In this case the photoreduction is mediated by the small equilibrium concentration of free flavin coenzyme present in a flavorprotein solution. Hence, it is suggested that flavoprotein reduction with EDTA as photosubstrate does not involve an excited state of the holoprotein, nor contact of EDTA with the enzyme, but exchange of electrons between enzyme flavin and free reduced flavin.

Light-mediated reduction of flavoproteins with flavins as catalysts.

The neutral flavosemiquinone has been studied in detail by electron spectroscopy. Isotopic (15N, 2H) and chemical substitutions have been carried out. A hyperfine coupling scheme of the neutral flavosemiquinone is described where N(5), N(10), CH3(10), CH3(8) and H(6) are involved. The highest spin density is located at N(5) as has also been found for the anionic and chelated form of the flavosemiquinone. The exchangeable proton in the neutral flavosemiquinone is attached to N(5) having a coupling constant of 7.6 G which is about the same magnitude as the coupling constant to N(5). The hyperfine couplings are interpreted in terms of spin densities and comparison is made with available quantum chemical calculations. Evidence is presented that two tautomeric forms of the neutral flavosemiquinone have to be considered which can be obtained in a pure form by suitable substitution of flavin derivatives. The two types of neutral flavosemiquinones are easily distinguishable by their electron spin resonance and light absorptions.

/pdf/the-chemical-and-electronic-structure-of-the-neutral-flavin-2ubpv5iqc1.pdf

The Chemical and Electronic Structure of the Neutral Flavin Radical as Revealed by Electron Spin Resonance Spectroscopy of Chemically and Isotopically Substituted Derivatives

Peter Hemmerich

Papers

Photoreduction of flavoproteins and other biological compounds catalyzed by deazaflavins.

Active-site probes of flavoproteins

High- and Low-mobility Populations of HP1 in Heterochromatin of Mammalian Cells

Light-mediated reduction of flavoproteins with flavins as catalysts.

The Chemical and Electronic Structure of the Neutral Flavin Radical as Revealed by Electron Spin Resonance Spectroscopy of Chemically and Isotopically Substituted Derivatives