Contribution of side-chain chromophores to the optical activity of proteins: model compound studies. I. Alpha-methyl-L-tyrosine

The optical rotatory power and the conformational energy of the amino acid α-methyl-L -tyrosine has been calculated as a function of molecular conformation. Comparison of the results of these theoretical calculations with experimental circular dichroism data indicates that the conformational freedom of this molecule is highly restricted. The most heavily populated conformations appear to be those near χ1 = 60°, χ2 = 80°, Ψ = 175°, and χ1 = 300°, χ2 = 80°, Ψ = 5°. The χ1 = 180° conformations are not likely to be populated to a significant extent at ordinary temperatures.     

Comparative studies of chromophore contents inside and outside the rhabdoms of arthropod compound eyes

Summary The intracellular distribution of visual pigments in the retinular cells of compound eyes of a butterfly (Papilio), a moth (Actias), a water scorpion (Ranatra), a dragonfly (Pantala), a crayfish (Procambarus), and a crab (Hemigrapsus) were investigated.The experiments measured the total surface area of rhabdom membrane in single compound eyes by light and electron microscopy, the densities of intramembrane particles in rhabdoms of compound eyes by freeze-fracture methods, and the quantities of chromophore molecules in compound eyes by high pressure liquid chromatography.Four species (moth, water scorpion, dragonfly, and crayfish) contain more than 75% of total chromophore probably as visual pigments in their rhabdoms, but the butterfly and the crab contain only 28.6% and 39.2%, respectively.The remainder of the chromophore molecules (Papilio 71.4%,Actias 5.7%,Ranatra 18.9%,Pantala 23.2%,Procambarus 24.3%, andHemigrapsus 60.8%) are supposed to exist mostly in the cytoplasm of the retinular cells. The localizing sites of such extrarhabdomeric chromophores are discussed in relation to the cytoplasmic membrane systems such as endoplasmic reticulum and lysosomal elements.In view of the results of the present experiment, it seems clear that the butterfly,Papilio, possesses a very large extra-rhabdomeric pool of chromophore that is available for rapid regeneration of visual pigment.Abbreviations HPLC high pressure liquid chromatography - ROR ratio of rhabdom to retinulae volume     

Determination of the secondary structure of proteins from circular dichroism spectra. IV. Contribution of aromatic amino acid residues into circular dichroism spectra of proteins in the peptide region

A new method for determination of the secondary protein structure from the CD spectra taking into account the contribution of aromatic amino acid residues is proposed. New proteins reference CD spectra for five secondary structures (alpha-helices, antiparallel and parallel beta-structures, beta-bends and irregular form) without contribution of aromatic residues are obtained. By means of this new method the secondary structure of sixteen different proteins was analysed. There is a good correlation of these results with the X-ray data.     

Antiproliferative activity and interactions with cell-cycle related proteins of the organotin compound triethyltin(IV)lupinylsulfide hydrochloride

Organotin compounds, particularly tri-organotin, have demonstrated cytotoxic properties against a number of tumor cell lines. On this basis, triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), a quinolizidine derivative, was synthesized and developed as a potential antitumor agent. This tin-derived compound exhibited potent antiproliferative effects on three different human cancer cell lines: teratocarcinoma of the ovary (PA-1), colon carcinoma (HCT-8) and glioblastoma (A-172). Cytotoxic activity was assessed by MTT and cell count assays during time course experiments with cell recovery after compound withdrawal. Significant cell growth inhibition (up to 95% in HCT-8 after 72 h of exposure), which also persisted after drug-free medium change, was reported in all the cell lines by both assays. In addition, the cytocidal effects exerted by IST-FS 29 appeared more consistent with necrosis or delayed cell death, rather than apoptosis, as shown by morphologic observations under light microscope, DNA fragmentation analysis and flow cytometry. In the attempt to elucidate whether this compound might affect genes playing a role in G1/S phase transition, the expressions of p53, p21(WAF1), cyclin D1 and Rb, mainly involved in response to DNA-damaging stress, were analyzed by Western blot. Heterogeneous patterns of expression during exposure to IST-FS 29 were evidenced in the different cell lines suggesting that these cell-cycle-related genes are not likely the primary targets of this compound. Thus, the present data seem more indicative of a direct effect of IST-FS-29 on macromolecular synthesis and cellular homeostasis, as previously hypothesized for other organotin complexes.     

Binding of quinoline analogues of echinomycin to deoxyribonucleic acid. Role of the chromophores.

Two novel antibiotics were isolated, designated compounds 1QN and 2QN respectively, having quinoline rings in place of one or both of the quinoxaline chromophores of echinomycin. Each removes and reverses the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs. For compound 1QN, the unwinding angle at I0.01 is almost twice that of ethidium, whereas for compound 2QN the value is indistinguishable from that of ethidium. Binding of both analogues produced changes in the viscosity of sonicated rod-like DNA fragments corresponding to double the helix extension found with ethidium, a feature characteristic of bifunctional intercalation by quinoxaline antibiotics. These results suggest that both compounds 1QN and 2QN behave as bifunctional intercalators but that compound 2QN produces only half the helix unwinding seen with compound 1QN and the natural quinoxalines. Binding curves for the interaction of both analogues with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis. Values for compound 2QN were also obtained by a fluorimetric method and found to agree well with the solvent-partition measurements. Compound 1QN bound most tightly to Micrococcus lysodeikticus DNA and, like echinomycin, exhibited a broad preference for (G + C)-rich DNA species. For compound 2QN no marked (G + C) preference was indicated, and the tightest binding among the natural DNA species studied was found with DNA from Escherichia coli. The two analogues also displayed different patterns of specificity in their interaction with synthetic nucleic acids. Compound 2QN bound to poly(dA-dT) slightly more tightly than to poly-(dG-dC), whereas compound 1QN displayed a large (approx. 11-fold) preference in the opposite sense. There was evidence of co-operativity in the binding to poly(dA-dT). It may be concluded that the chromophore moieties play an active role in determining the capacity of quinomycin antibiotics to recognize and bind selectively to specific sequences in DNA.     

The dominant role of side-chain backbone interactions in structural realization of amino acid code. ChiRotor: a side-chain prediction algorithm based on side-chain backbone interactions

The basic differences between the 20 natural amino acid residues are due to differences in their side-chain structures. This characteristic design of protein building blocks implies that side-chain-side-chain interactions play an important, even dominant role in 3D-structural realization of amino acid codes. Here we present the results of a comparative analysis of the contributions of side-chain-side-chain (s-s) and side-chain-backbone (s-b) interactions to the stabilization of folded protein structures within the framework of the CHARMm molecular data model. Contrary to intuition, our results suggest that side-chain-backbone interactions play the major role in side-chain packing, in stabilizing the folded structures, and in differentiating the folded structures from the unfolded or misfolded structures, while the interactions between side chains have a secondary effect. An additional analysis of electrostatic energies suggests that combinatorial dominance of the interactions between opposite charges makes the electrostatic interactions act as an unspecific folding force that stabilizes not only native structure, but also compact random conformations. This observation is in agreement with experimental findings that, in the denatured state, the charge-charge interactions stabilize more compact conformations. Taking advantage of the dominant role of side-chain-backbone interactions in side-chain packing to reduce the combinatorial problem, we developed a new algorithm, ChiRotor, for rapid prediction of side-chain conformations. We present the results of a validation study of the method based on a set of high resolution X-ray structures.     

Photochemical and chemical studies on the chromophore of bacteriorhodopsin.

The chromophore (purple complex) of bacteriorhodopsin is reduced by sodium borohydride upon illumination to RPhv with a three-peaked absorption band at 360 nm. Treatment of this reduction product with ultraviolet light or acid yields a modified product from which retro-retinyllysine can be obtained by alkaline hydrolysis. No reduction of the 412 nm complex was found. Under specific conditions the purple complex equilibrates with a photochemically active 460 nm form that can be reduced by borohydride in the dark. This reduction product RP460 behaves idential to RPHV. Reconstitution of the purple complex from chromophore-free membrane (apomembrane) and retinal occurs via intermediates. The first (lambdamax 400nm) shows a three-peaked absorption band and is reduced to RP400 without a change of the three-peaked absorption (lambdamax 360 nm). The same product is obtained from apomembrane and retinol. Detergents shift the absorption band to 330 nm in all cases. From the experiments described no participation of retro-retinal structures during the photochemical cycle can be concluded but stereospecific interaction of the retinal moiety with the protein resulting in a specific retinal conformation os omdocated by the spectral changes observed.     

Contribution to the probabilistic theory of neural nets: IV. Various models for inhibition

Input-output formulas are derived for a neuron upon which converge single axones of two other neurons, which are subjected to a Poisson shower, where a number of different assumptions are made concerning the mechanism of inhibition. In one assumption so-called “bilateral pre-inhibition” is considered. That is to say, both neuronsN ₁ andN ₂ may exciteN ₃, but if the stimulus of one of them follows within a certain interval σ of the other, the second stimulus is not effective. This model is essentially no different from that involving two excitatory neurons acting upon a neuron having a refractory period. Another mechanism considered involves so-called “pre-and-post” inhibition, in which if two stimuli fromN ₁ andN ₂ fall within σ,both are ineffective. This case being mathematically much more involved than the preceding, an approximation method is used for deriving the input-output formula. Previous papers of this series are denoted by I, II, and III in this paper.     

Model studies of interactions between nucleic acids and proteins: hydrogen bonding of amides with nucleic acid bases.

The formation of hydrogen bonded complexes between nucleic acid bases and acetamide has been studied by nuclear magnetic resonance in CDC13 at different temperatures. Pairs of hydrogen bonds are formed when acetamide binds to nucleic acid bases. Thermodynamic parameters have been computed and compared to those obtained for the association of carboxylic acids with nucleic acid bases. The role of hydrogen bonded complexes in the association of proteins with nucleic acids is discussed.     

Microsequence analysis of peptides and proteins: IV. Structural studies on human leukocyte interferons

The sequence of the tryptic peptides of three major species of human leukocyte interferon was determined by microsequencing procedures. The peptides were aligned by comparison with the amino acid sequences predicted by the DNA sequences of recombinants containing leukocyte interferon-coding inserts. In addition, extended NH₂-terminal amino acid sequences of two human leukocyte interferons produced in Escherichia coli by recombinant DNA methodology are also reported. This report demonstrates application of microsequencing methodology to low nanomole and subnanomole amounts of proteins and peptides of biological interest.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Chlorination studies. IV. The reaction of aqueous hypochlorous acid with pyrimidine and purine bases

The labile intermediates and stable end products formed by the reaction of aqueous HOCl with thymine, uracil, 5-Br-uracil, N,N-dimethyluracil and 6-methyluracil have been identified. The purine ring system of guanine, adenine and xanthine was more resistant to attack by aqueous HOCl and reaction times of one week resulted in the formation of parabanic acid. Caffeine and theophylline under similar reaction conditions yielded N,N-dimethylparabanic acid.