Response time and threshold of a random net

The response time of a random net is defined as the expected time (measured in the number of synaptic delays) required for the excitation in the net (measured by the fraction of neurons firing per unit time) to reach a certain level. The response time is calculated in terms of the net parameters as a function of the intensity of the outside stimulation. Two principal types of cases are studied, 1) an instantaneous initial stimulation, and 2) continuously applied stimulation. It is shown that for a certain type of net where the required level of excitation is small, the response time-intensity equation reduces to the one derived on the basis of the “one-factor” theory applied to a neural connection. More general assumptions, however, give different types of equations. The concept of the “net threshold” is defined, and its calculation indicated. The net threshold for instantaneous stimulation is, in general, greater than that for continuous stimulation. The results are discussed with reference to existing theories of reaction times.     

Self-assembly of biological macromolecules.

The genetic apparatus of the cell is responsible for the accurate biosynthesis of the primary structure of macromolecules which then spontaneously fold up and, in certain circumstances, aggregate to yield the complex tertiary and quaternary structures of the biologically active molecules. Structures capable of self-assembly in this range from simple monomers through oligomers to complex multimeric structures that may contain more than one type of polypeptide chain and components other than protein. It is becoming clear that even with the simpler monomeric enzymes there is becoming clear that even with the simpler monomeric enzymes there is a kinetically determined pathway for the folding process and that a folded protein must now be regarded as the minimum free energy form of the kinetically accessible conformations. It is argued that the denatured subunits of oligomeric enzymes are likely to fold to something like their final structure before aggregating to give the native quaternary structure and the available evidence would suggest that this is so. The importance of nucleation events and stable intermediates in the self-assembly of more complex structures is clear. Many self-assembling structures contain only identical subunits and symmetry arguments are very successful in accounting for the structures formed. Because proteins are themselves complex molecules and not inelastic geometric objects, the rules of strict symmetry can be bent and quasi-equivalent bonding between subunits permitted. This possibility is frequently employed in biological structures. Conversely, symmetry arguments can offer a reliable means of choosing between alternative models for a given structure. It can be seen that proteins gain stability by growing larger and it is argued in evolutionary terms that aggregation of subunits is the preferred way to increase the size of proteins. The possession of quaternary structure by enzymes allows conferral of other biologically important properties, such as cooperativity between active sites, changes of specificity, substrate channelling and sequential reactions within a multi-enzyme complex. Comparison is made of the invariant subunit compositions of the simpler oligomeric enzymes with the variation evidently open to, say, the 2-oxoacid dehydrogenase complexes of E. coli. With viruses, on the other hand, the function of the quaternary structure is to package nucleic acid and, as an example, the assembly and breakdown of tobacco mosaic virus is discussed. Attention is drawn to the possible ways in which the principles of self-assembly can be extended to make structures more complicated than those that can be formed by simple aggregation of the comonent parts.     

Self-assembly of membrane junctions.

We present a mechanism for the aggregation of mobile intermembrane junctions, such as the connexon dyad of gap junctions. The model demonstrates that intermembrane repulsion provides a powerful self-assembly pressure. If the membrane repulsion is strong enough to prevent membrane adhesion, then the self-assembly pressure is of effective infinite range.     

Self-assembly of extracellular tubular structures of non-protein nature produced by an actinomycete]

When grown on the solid synthetic medium with glucose as the only carbon source the dedifferentiated "fructose" mutant of Actinomyces roseoflavus var. roseofungini accumulated aggregates of tubular-like structures. The individual tubules had the internal diametre of 80 A and external diametre of approximately 200-220 A. These structures were isolated as a distinct fraction and their non-protein nature was demonstrated. They were easily soluble in acetone and reconstitutable in vitro. The possible significance of production of self-assembling structures by a mutant with impaired differentiation is discussed. The possibility of involvement of self-assembly processes in the formation of surface sheath of aerial mycelium in normally differentiating actinomycetes is mentioned.     

Reduction to below threshold levels of glycolate oxidase activities in transgenic tobacco enhances photoinhibition during irradiation

The effects of decreased flux in the glycolate pathway on photoinhibition was investigated in transgenic tobacco (Nicotiana tabacum L. cv. SR1) plants. These plants harbored a pumpkin cDNA for glycolate oxidase (GO), an enzyme in the glycolate pathway, under the control of the cauliflower mosaic virus 35S promoter. Some transformants showed both reduced amounts and reduced activities of GO. The decrease of GO was enhanced at a later growth stage of these transformants, whereas no changes were observed in the amounts of other enzymes in the glycolate pathway, such as hydroxypyruvate reductase and serine glyoxylate aminotransferase. The phenotype grown under a low light condition (30 microE s(-1) m(-2)) resembled that of the wild type. Transformants with about 35% lower GO activity than wild type, had a lower Fv/Fm under 500 microE s(-1) m(-2) irradiation for 8 h. After 60 microE s(-1) m(-2) irradiation for 8 h, Fv/Fm was lowered in some transformants with less than 20% of the GO activity of the wild type. These results suggest that photosynthesis was susceptible to photoinhibition with reduction to below threshold levels of GO activities and that higher activities of GO are required under a higher irradiation. The increase in the electron transport rate (ETR) with increased irradiation was suppressed only in transformants that had GO activity one-third less than the wild type, suggesting that the regeneration of the substrate for the Calvin cycle was decreased only when there was an extreme reduction of GO. These results also suggest that the photosystem was disturbed when the concentration of the substrate for the Calvin cycle decreased until it became insufficient to receive the excess photon energy generated in each light environment.     

Elucidation of a novel polypeptide cross-link involving 3-hydroxykynurenine.

3-Hydroxykynurenine, a metabolite of tryptophan, is a powerful antioxidant and neurotoxin. The neurotoxicity results from the oxidation of 3-hydroxykynurenine, and hydroxyl radicals, formed via H(2)O(2), may also be implicated [Okuda, S., Nishiyama, N., Saito, H. , and Katsuki, H. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12553-12558]. Oxidation of o-aminophenols, such as 3-hydroxykynurenine, also results in the formation of highly reactive quinonimines. Thus, one possible consequence of 3-hydroxykynurenine oxidation may be covalent modification of cellular macromolecules. Such a process could contribute to the neurotoxicity and may potentially be important in other tissues, such as the human lens, where 3-hydroxykynurenine functions as a UV filter. In this work, we demonstrate that 3-hydroxykynurenine can bind to protein amino groups and, further, that under oxidative conditions, 3-hydroxykynurenine can function to cross-link polypeptide chains. The structure of the cross-linked moiety, using the peptide glycyllysine, has been elucidated. The cross-link, which is both colored and fluorescent, involves the peptide alpha-amino groups. Proteins modified by 3-hydroxykynurenine become colored and fluorescent as well as cross-linked. LC-MS studies indicate that the cross-link is also present in gamma-crystallin, following incubation of this lens protein in the presence of 3-hydroxykynurenine. Similar posttranslational modifications of lens proteins accompany cataract formation, and knowledge of the precise mode of reaction of 3-hydroxykynurenine with proteins will assist in determining if 3-hydroxykynurenine is involved in degenerative conditions in which oxidation of such aminophenols is implicated.     

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.     

Folding of the villin headpiece subdomain from random structures. Analysis of the charge distribution as a function of pH

The structure of the 36 residue villin headpiece subdomain is investigated with the electrostatically driven Monte Carlo method. The ECEPP/3 (Empirical Conformational Energy Program for Peptides) force field, plus two different continuum solvation models, were used to describe the conformational energy of the chain with both blocked and unblocked N and C termini. A statistical analysis of an ensemble of ab initio generated conformations was carried out, based on a comparison with a set of ten native-like structures derived from published experimental data, by using rigid geometry and NMR-derived constraints obtained at pH 3.7. The ten native-like structures satisfy the NMR-derived constraints. The whole ensemble of conformations of the terminally unblocked villin headpiece sub-domain, generated by using ECEPP/3 with a continuum solvation model, were subsequently evaluated at pH 3.7 with a potential function that includes ECEPP/3 combined with a fast multigrid boundary element method. At pH 3.7, the lowest-energy conformation found during the conformational search satisfies approximately 70% of both the distance and the dihedral-angle constraints, and possesses the characteristic packing of three phenylalanine residues that constitute the main part of the hydrophobic core of the molecule. On the other hand, computations at pH 3.7 and pH 7.0 for the ten native-like structures satisfying the NMR-derived constraints indicate a substantial change in the charge distribution for each type of amino acid residue with the change in pH. The results of this study provide a basis to understand the effect of the interactions, such as hydrophobicity, charge-charge interaction and solvent polarization, on the stability of this small alpha-helical protein.     

The membrane skeleton of erythrocytes. A percolation model.

The spectrin network on the cytoplasmic surface of the erythrocyte membrane is modeled as a triangular lattice of spectrin tetramers. This network obstructs lateral diffusion of proteins and provides mechanical reinforcement to the membrane. These effects are treated in a systematic and unified manner in terms of a percolation model. The diffusion coefficient is obtained as a function of the fraction of normal spectrin tetramers for both static and fluctuating barriers. The elasticity of the network is calculated as a function of the fraction of normal spectrin and the ratio of bending to stretching energies. For static barriers, elasticity and lateral diffusion are incompatible: if a network is connected enough to be elastic, it is connected enough to block long-range lateral diffusion. The elasticity and the force required for mechanical breakdown go to zero at the percolation threshold; experimental evidence suggests the existence of a stability threshold at or near the percolation threshold. The model is qualitatively applicable to other cells with membrane skeletons, such as epithelial cells, in which localization of membrane proteins is essential to differentiation.     

Self-assembly of DNA-polymer complexes using template polymerization.

The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles.     

Reactions of the protein radical in peroxide-treated myoglobin. Formation of a heme-protein cross-link

Reaction of horse myoglobin with H2O2 oxidizes the iron to the ferryl (Fe(IV) = O) state and produces a protein radical that is rapidly dissipated by poorly understood mechanisms. As reported here, the reaction with H2O2 results in covalent binding of up to 18% of the prosthetic heme group to the protein. The chromophore of the protein-bound prosthetic group is very similar to that of heme itself. High performance liquid chromatography of tryptic digests indicates that the formation of heme-bound peptides is associated with disappearance of the peptide with the sequence YLE-FISDAIIHVLHSK corresponding to residues 103-118 of horse myoglobin. Amino acid analysis, terminal amino acid sequencing, and liquid secondary ion mass spectrometry establish that the heme is primarily attached to this peptide. The heme appears to be bound to the tyrosine residue because the tyrosine is the only amino acid that disappears from the amino acid analysis. The mass spectrometric data indicates that the heme-peptide is formed without addition or loss of an oxygen or other major structural fragment. The site of attachment to the heme group has not been unambiguously determined, but the heme vinyl groups are not essential for the reaction because equal cross-linking is observed in H2O2-treated mesoheme-reconstituted myoglobin. The results are most consistent with binding of tyrosine 103 to a meso-carbon of the prosthetic heme group.     

Reversible denaturation of disulfide-reduced ovalbumin and its reoxidation generating the native cystine cross-link.

The authors in a previous report (Klausner, R. D., Kempf, C., Weinstein, J. N., Blumenthal, R., and van Renswoude, J. (1983) Biochem. J. 212, 801-810) have argued that native folding of ovalbumin occurs during translation, but not in a renaturation system of the denatured form. To re-examine the possibility, we searched for the conditions of correct oxidative refolding of denatured disulfide-reduced ovalbumin. Data of trypsin resistance, CD-spectrum, and selective reactivity of cysteine sulfhydryls revealed that the fully denatured protein can refold into the native conformation under disulfide-reduced conditions. The interconversion between the native and denatured forms was fully reversible with a free energy change for unfolding of 6.6 kcal/mol at 25 degrees C. Subsequent reoxidation under a variety of redox conditions generated only one disulfide bond in the reduced refolded protein with six cysteine sulfhydryls. Furthermore, the regenerated disulfide was found by peptide analyses to correspond to the native disulfide pairing, Cys73-Cys120. We, therefore, concluded that co-translational folding, if any, is not requisite for the correct oxidative folding of ovalbumin.     

Wavelength dependence for the photoreversal of a psoralen-DNA cross-link

We report an action spectrum for the photoreversal of a psoralen cross-link joining two self-complementary DNA oligonucleotides. The cross-link was formed between two thymines (T) on opposite strands of the DNA oligomers and 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT). For comparison, we also present an action spectrum for the photoreversal of the isolated diadduct T-HMT-T. The wavelength dependence for the diadduct photoreversal parallels its absorption spectrum. Both the diadduct and the cross-linked DNA can be photoreversed by exposure to light with wavelengths between 240 and 313 nm. We did not observe photoreversal at 334 nm or above. At least two distinct absorption bands appear to contribute to photoreversal. We measured a quantum yield of 0.16 for photoreversal of the isolated diadduct at wavelengths between 240 and 266 nm. For wavelengths above 280 nm, the quantum yield is 0.30. We also observed a preferential photoreversal at the furan end of the psoralen in the T-HMT-T diadduct. In contrast, the cross-linked DNA oligonucleotides preferentially photoreversed at the pyrone end of the psoralen adduct. The rate constant for photoreversal of the cross-linked DNA is larger than that for the isolated diadduct at wavelengths below 300 nm.