Showing papers in "Biochemistry in 1974"

Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins.

Abstract: The helix, s Applequist, 1963) in which the Zimm-Bragg parameters u and s are defined respectively as the cooperativity factor for helix initiation, and the equi- librium constant for converting a coil residue to a helical ~~~~

Intermolecular energy transfer in the bioluminescent system of Aequorea

Abstract: The jellyfish Aequoreci emits green light whereas the photoprotein aequorin extracted from the same organism emits blue light when Ca2+ is added. Because the photogenic cells contain a green fluorescent protein (GFP) in addition to aequorin, an energy transfer from the light emitter of aequorin to GFP has been postulated. In the present study, GFP has been purified, crystallized, and partially characterized and an energy transfer in citro from aequorin to this protein has been demonstrated. GFP was found to consist of several kinds of isomeric proteins, of which two kinds predominated. After separation, both kinds evinced the same absorption maxima T he bioluminescent jellyfish Aequorea emits "green light" in cico, whereas the pure photoprotein aequorin extracted from the same organism emits "blue" light on addition of Ca?-. This difference, along with some of the properties of the purified aequorin and also the occurrence of a green fluores- cent protein in the luminescent extract, was first reported over 10 years ago (Shimomura et al., 1962). The blue light (A,,,,, 470 nm) of aequorin (mol wt ca. 30,000; Shimomura and Johnson, 1969; Kohama et a/., 1971) results from an intramolecular reaction when Ca2+ is added, yielding a reaction product "BFP" ("blue fluorescent protein") which shows a fluorescence spectrum corresponding exactly to the bioluminescence spectrum (Shimomura and Johnson, 1970a). The chemical structure of the light emitting chromophore has been recently determined to be 2-(p-hydroxyphenylacetyl)- atnino-3-benzyl-5-(p-hydroxyphenyl)pyrazine (Shimomura

Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6-A resolution.

Abstract: The complex of porcine trypsin with soybean tryp- sin inhibitor (Kunitz) was crystallized from 17% ethanol at pH 7. The crystals have P212121 symmetry with a = 59.0, b = 62.2, and c = 150.5 A. They contain one molecule of the 1:l complex in the crystallographic asymmetric unit. Three-dimen- sional X-ray diffraction data were measured for these crystals and for crystals soaked with 0.3 mM K2PtC14, 1 mM K3UOzF5, 5 mM mercury salicylate, 0.2 mM (NH4)3IrC16, and 5 mM mercury acetate. All strong reflections to 2.6 A were measured, including almost all reflections to 3.8 A and 56% of the reflec- tions between 3.0 and 2.6 A. Beyond 3.8 A only the K3U02F5 and salicylate derivatives were useful. In the resulting electron density maps the trypsin molecule was easily identifiable, and although some parts of the inhibitor were not clear, a precise interpretation of residues 1'-93' was made, including the con- tact region. Coordinates for this part of the inhibitor, and for adjacent parts of the trypsin molecule, were refined by the real-

Fluorescence and optical characteristics of reduced flavines and flavoproteins.

Abstract: Publisher Summary This chapter discusses the fluorescence and optical characteristics of reduced flavins and flavoproteins. Flavins and flavoproteins in their oxidized state (Flex) are typically yellow, with the absorption spectrum in the visible and near-ultraviolet (UV) range being characterized, by two well-resolved bands centered around 450 and 370 nm. This chromophore, free in solution, exhibits a strong fluorescence that is maximal around 520 nm; when the chromophore is bound to flavoproteins, its emission can be fully quenched. Studies show that the near-UV absorption spectrum of the 4a,5-dihydroflavin chromophore is similar, in its appearance, to that of its 1,5-dihydro isomer, but its shape is even more dependent on temperature and solvent. In particular, substituents that have an effect on the internal mobility of the molecule were found to cause major spectral changes on the chromophore free in solution and also when bound to various apoflavoproteins. At ambient temperature, 4a,5-dihydroflavins show a weak fluorescence only when an appropriate substituent decreases their internal mobility. However, at low temperature in rigid ethanol glasses or when bound to apoflavoproteins, 4a,5-dihydroflavins exhibit a rather strong fluorescence emission maximal in the range 480-530 nm.

Partial specific volumes and interactions with solvent components of proteins in guanidine hydrochloride.

Abstract: The partial specific volumes of twelve proteins were determined by density measurements. For these proteins in their native state, the determined values of partial specific volumes are generally in good agreement with the accepted literature values except in the case of bovine a-lactalbumin. The determined value of 0.704 is similar to that found for lysozyme. Preferential interaction parameters of 6 M guanidine hydrochloride with these proteins were also measured. For the twelve proteins studied, the preferential interaction with solvent components vanes between 0 and 0.17 g of guanidine A lthough an exact knowledge of the partial specific volume, 6, of a protein is essential for the determination of molecular weights from ultracentrifuge data and small-angle X-ray scattering, in the past this parameter was seldom measured. The molecular weights were calculated from values of 6 assumed, or calculated from amino acid composition. And yet the importance of accurate measurements of the partial specific volume was generally recognized, since a small error in that parameter is multiplied several fold in the calcula- tion of the molecular weight, in particular when measurements are carried out in concentrated solutions of denaturant, such as 6 M Gdn.HCl,I a frequent practice in studies of subunit systems. Uncertainties in estimates of molecular weight can lead to wrong conclusions about the number of subunits in the native macromolecular assembly and to serious errors in the calculation of the thermodynamic parameters of associating systems. A classical example is the uncertainty which prevailed for several years about the exact .number of polypeptide chains in rabbit muscle aldolase (Kawahara and Tanford, 1966; Schachman and Edelstein, 1966; Castellino and Barker, 1968; Reisler and Eisenberg, 1969 ; Meighen and Schachman, 1970). Until recently, the methods available for the measurement of the partial specific volume either required frequently prohibi- tive amounts of material, as in conventional pycnometry, or involved long complicated procedures, as in the density gradient column technique (Linderstrgm-Lang and Lanz, 1935; Hvidt ef al., 1954; Reithel and Sakura, 1963). Recently, several new and elegant approaches to the measurement of 8, requiring small amounts of material, have been described. These include the HD-D20 method of Edelstein and Schach- man (1967), the magnetic float method of Ulrich et af. (1964), and the precision densimeter, based on the frequency of vibra- tion of a "tuning fork," consisting of a sample-filled quartz hydrochloride per g of protein. In no case is interaction preferential with water. The total binding of denaturant to protein is calculated and the correlation between the observed and expected number of denaturant molecules bound is found to be good, if peptide bonds and aromatic side chains are taken as the binding sites. The changes in volume upon transfer from dilute salt to 6 M guanidine hydrochloride of the proteins studied are calculated and compared with theoretical values reported in the literature.