The hydration of protein secondary structures

The hydration of the main-chain carbonyl (CO) groups in proteins have been studied using infra-red spectroscopy, and computer-graphics analysis of high resolution protein crystal structures. The IR measurements indicate that the strength of water binding to the CO groups is lower in beta-sheet proteins compared with alpha-helical ones. Analysis of the protein crystal structures shows that this is due primarily to differences in the geometry of water-CO group interactions in the two types of secondary structure.     

Circadian rhythms in the green sunfish retina

We investigated the occurrence of circadian rhythms in retinomotor movements and retinal sensitivity in the green sunfish, Lepomis cyanellus. When green sunfish were kept in constant darkness, cone photoreceptors exhibited circadian retinomotor movements; rod photoreceptors and retinal pigment epithelium (RPE) pigment granules did not. Cones elongated during subjective night and contracted during subjective day. These results corroborate those of Burnside and Ackland (1984. Investigative Ophthalmology and Visual Science. 25:539-545). Electroretinograms (ERGs) recorded in constant darkness in response to dim flashes (lambda = 640 nm) exhibited a greater amplitude during subjective night than during subjective day. The nighttime increase in the ERG amplitude corresponded to a 3-10-fold increase in retinal sensitivity. The rhythmic changes in the ERG amplitude continued in constant darkness with a period of approximately 24 h, which indicates that the rhythm is generated by a circadian oscillator. The spectral sensitivity of the ERG recorded in constant darkness suggests that cones contribute to retinal responses during both day and night. Thus, the elongation of cone myoids during the night does not abolish the response of the cones. To examine the role of retinal efferents in generating retinal circadian rhythms, we cut the optic nerve. This procedure did not abolish the rhythms of retinomotor movement or of the ERG amplitude, but it did reduce the magnitude of the nighttime phases of both rhythms. Our results suggest that more than one endogenous oscillator regulates the retinal circadian rhythms in green sunfish. Circadian signals controlling the rhythms may be either generated within the eye or transferred to the eye via a humoral pathway.     

N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.     

Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy

The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]-and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13C]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., & Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Helix geometry in proteins

In this report we describe a general survey of all helices found in 57 of the known protein crystal structures, together with a detailed analysis of 48 alpha-helices found in 16 of the structures that are determined to high resolution. The survey of all helices reveals a total of 291 alpha-helices, 71 3(10)-helices and no examples of pi-helices. The conformations of the observed helices are significantly different from the "ideal" linear structures. The mean phi, psi angles for the alpha- and 3(10)-helices found in proteins are, respectively, (-62 degrees, -41 degrees) and (-71 degrees, -18 degrees). A computer program, HBEND, is used to characterize and to quantify the different types of helix distortion. alpha-Helices are classified as regular or irregular, linear, curved or kinked. Of the 48 alpha-helices analysed, only 15% are considered to be linear; 17% are kinked, and 58% are curved. The curvature of helices is caused by differences in the peptide hydrogen bonding on opposite faces of the helix, reflecting carbonyl-solvent/side-chain interactions for the exposed residues, and packing constraints for residues involved in the hydrophobic core. Kinked helices arise either as a result of included proline residues, or because of conflicting requirements for the optimal packing of the helix side-chains. In alpha-helices where there are kinks caused by proline residues, we show that the angle of kink is relatively constant (approximately 26 degrees), and that there is minimal disruption of the helix hydrogen bonding. The proline residues responsible for the kinks are highly conserved, suggesting that these distortions may be structurally/functionally important.     

Influence of Drought Acclimation and CO(2) Enrichment on Osmotic Adjustment and Chlorophyll a Fluorescence of Sunflower during Drought

Osmotic adjustment occurred during drought in expanded leaves of sunflowers (Helianthus annuus var Hysun 30) which had been continuously exposed to 660 microliters CO₂ per liter or had been previously acclimated to drought. The effect was greatest when the treatments were combined and was negligible in nonacclimated plants grown at 340 microliters CO₂ per liter. The concentrations of ethanol soluble sugars and potassium increased during drought but they did not account for the osmotic adjustment. The delay in the decline in conductance and relative water content and in the loss of structural integrity with increasing drought was dependent on the degree of osmotic adjustment. Where it was greatest, conductance fell from 5.8 millimeters per second on the first day of drought to 1.3 millimeters per second on the fourth day and was at approximately the same level on the eighth day. The relative water content remained constant at 85% for three days and fell to 36% on the sixth day. There was no evidence of leaf desiccation even on the eighth day. In contrast, the conductance of leaves showing minimal adjustment fell rapidly after the first day of drought and was negligible after the fourth, at which time the relative water content was 36%. By the sixth day of drought, areas near the margins of the leaves were desiccating and the plants did not recover upon rewatering. Despite the differences in the rate of change of conductance and relative water content during drought, photosynthetic electron transport activity, inferred from measurements of chlorophyll a fluorescence in vivo and PSII activity of isolated thylakoids, remained functional until desiccation occurred.     

Purification of proteins from polyacrylamide gels, free of detergent or dye

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.     

Polar hydrogen positions in proteins: Empirical energy placement and neutron diffraction comparison

A method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X-ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.     

Differential localization of leu-enkephalin and hyperglycemic hormone in axon terminals of the sinus gland of the crabsCarcinus maenas,eriocheir sinensis,andUca pugilator

Summary Leu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.