Coprecipitation of thermal lysine-rich proteinoids with polyribonucleotides.

Major variables in interactions between basic thermal proteinoids and homopolyribonucleotides were magnesium concentration in solution (0–40 mM) and mol% lysine in the proteinoid (16–55%). The formation of microparticles was monitored both by the turbidity and by the mass of precipitate formed. Under some conditions, only, was the turbidity reading a reliable indication of the amount of precipitate. Increasing concentration of Mg²⁺ tended to displace proteinoid from the complex with polynucleotide. Of 4 polynucleotides, only polyguanylic acid showed an enhanced precipitation of proteinoid in the presence of Mg²⁺, and then only with those having high lysine contents. At high lysine contents, the amount of proteinoid in the precipitate was inversely proportional to the lysine content of the proteinoids, probably due to decreased sidechain interactions. The precipitation with polynucleotides is partly a function of the amino acid composition of the proteinoid; therefore the interaction of thermal proteinoids with polynucleotides appears to be a tool that can be used to study specificities of interactions between proteins and nucleic acids.     

Natural self-organization of polynucleotides and polypeptides in protobiogenesis: Appearance of a protohypercycle

Basic thermal polyamino acids or proteinoids have been reported to be catalytic for both self-instructing polymerization of amino acids and internucleotide synthesis. We show theoretically that a complex suspension of thermal proteinoids, free amino acids, nucleotides and ATP as an energy source can exhibit an evolutionary character. The suspension can produce a prototype of Eigen's hypercycle, or protohypercycle, for which translation proceeds from amino acid to nucleotide. The protohypercycle is suggested to be an evolutionary precursor of the hypercycle, in which translation is from nucleotide to amino acid. The possibility that the Fox-Nakashima microsphere containing both lysine-rich and acidic proteinoids may work as a model of a protohypercycle is considered.     

Proteinoids as complexes of polyamino acids with melanoidins

Proteinoids have been demonstrated as complexes of amino acid polymers with melanoidin pigments. Some physico-chemical properties of proteinoid pigments were studied in comparison with the standard melanoidins. Proteinoid pigments were able to enhance oxidoreduction and hydrolysis reactions, and their activity was comparable with the activity of the corresponding polyamino acid components or even of the entire proteinoids. The pigmented proteinoids had relatively strong ESR signal indicating the presence of free radicals into melanoidin components. Hypothetical participation of proteinoid melanoidin pigments in prebiotic evolution is discussed.     

Excitable cell made of thermal proteinoids

Such phenomena as electrical polarization across the membrane, electrical discharges, current-voltage characteristics, negative resistance and some light characteristics are described for synthetic cells made of thermal proteinoids. The thermal proteinoid cell is considered as a structural and functional model of the excitable natural cell.     

Porphyrin-proteinoid complexes as models of prebiotic photosensitizers

Photochemical activity as well as some spectral properties of various porphyrins and their model complexes with proteinoids were studied. Photochemical activity increased from the less advanced biosynthetic chlorophyll precursors to the more advanced compounds. The significant increase was detected as a result of the formation of complexes of phytol-containing porphyrins with proteinoids, which brings about the disaggregation of the pigment clusters formed in the aqueous environment. Hemin is photochemically inactive, either alone or in complex with proteinoid. Although hemoproteinoid was demonstrated to be able to catalyse photochemical electron transfer, its activity is about 20 times lower when compared with the chlorophyll a-proteinoid complex. Experimental results are discussed in context of the early evolution of photosynthesis.     

A model for the origin of stable protocells in a primitive alkaline ocean

When a mixture of the eighteen proteinous amino acids are suitably heated in the dry state with seawater salts, a copolyamino acid results. One fraction of this polymer is found, through isoelectric focusing, to consist of a mixture of acidic and basic proteinoids, each of sharply limited heterogeneity. When one fraction of the seawater proteinoid is dissolved in hot water, and the solution is cooled, proteinoid microspheres result. These have properties in common with simpler types, but are also stable at pH values to 9, in common with microspheres prepared by mixing acidic and basic proteinoids. These processes thus constitute a simple model for the origin of a protocell stable in a primitive alkaline ocean.     

Hypotheses on the establishment of a genetic code and transfer of information from proteinoids to nucleic acids

An hypothesis is proposed in which the specificity of interaction between an aminoacid and a nucleotide sequence of a tRNA would be enhanced by a ternary association with a specific proteinoid. These strict relations would have led to the present genetic code that we know. It is also proposed that the origin of the enzymatic activity of the primitive proteinoids would have arisen from the presence of different substrates during polymerisation, which would have favored specific sequences of aminoacids by forming more stable complexes with them, corresponding to the lowest free enthalpy. The information included in the aminoacid sequences of the proteinoids would have been transferred to messenger type RNA, according to a mechanism reverse of that for the present process for protein synthesis, and then to DNA.     

Formation of proteinoid microspheres under simulated prebiotic atmospheres and individual gases.

The formation of microspheres from acidic and basic proteinoids was attempted under simulated prebiotic atmospheres and constituent gases thereof. Both types of proteinoid yielded microspheres under carbon dioxide, carbon monoxide, methane, hydrogen sulfide, hydrogen, nitrogen, and oxygen (tested separately) and also under nitrogen-carbon dioxide atmospheres; higher proportions of carbon dioxide resulted in fewer spheres from basic proteinoid. Neither type of proteinoid formed spheres on 10-minute exposure to ammonia or methane-hydrogen-ammonia atmospheres. (Brief exposure resulted in spheres from basic proteinoid.) The effects, both qualitative and quantitative, were indicated by control experiments to be due to pH, rather than to the specific gas (or ion). The results suggest that the proteinoid microsphere model for protocells is applicable under a variety of possible prebiotic atmospheres, with some restrictions imposed by pH.     

Proteinoid microspheres more stable in hot than in cold water

The occurrence of organisms of primitive appearance near submarine hydrothermal vents has indicated sea-floor conditions that are like those under which proteinoid microspheres are produced in the laboratory. Experimental examination of the question of whether some proteinoid microspheres might be stable in hot water has revealed proteinoids that are soluble in cold water but precipitate on heating. Unanswered questions are discussed.     

Genetic code correlations: Differential rates of non-enzymatic activation of hydrophobic amino acids by ATP

The relative rates of non-enzymatic activation of several hydrophobic amino acids by ATP have been found to bear an interesting relationship to the ordering of these amino acids in the genetic anticode. All of these hydrophobic amino acids (phe, leu, val, ile and met) have adenylic acid, the most hydrophobic nucleotide, as the central and most important member of their anticodons, and the ordering of their relative rates of nonenzymatic activation by ATP has been found not to correlate with the ordering of the hydrophobicities of the amino acids themselves, but rather with the ordering of the average estimates of the hydrophobicities of their respective anticodonic dinucleotides. These data suggest that the genetic code is based not just on hydrophobic relationships or affinities between amino acids and nucleotides, but perhaps more importantly, on the total reaction chemistry between amino acids and nucleotides.     

Assembly of microspheres from acidic proteinoids and histones or histone-like proteinoids

Basic polyamino acids, whether organismic or synthetic in origin, alter the morphology of proteinoid microspheres in which they are included. The particles obtained with histone are essentially indistirguishable under the light microscope from those obtained with proteinoids of histone-like composition (histonoids). In each instance, structures of morphological complexity similar to those of some microfossils are obtained.     

Rationalization of some genetic anticodonic assignments

The genetic code appears to be a logic matrix in which, generally speaking, there is a correlation between the hydrophobicities of amino acids and their anticodonic nucleotides. There are several exceptions to this generality, however, and using previous data on hydrophobicity and binding constants, coupled with new data on reaction rates, we rationalize several of the anticodonic assignments.     

tRNA discrimination by T. thermophilus phenylalanyl-tRNA synthetase at the binding step

The extent of tRNA recognition at the level of binding by Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS), one of the most complex class II synthetases, has been studied by independent measurements of the enzyme association with wild-type and mutant tRNA(Phe)s as well as with non-cognate tRNAs. The data obtained, combined with kinetic data on aminoacylation, clearly show that PheRS exhibits more tRNA selectivity at the level of binding than at the level of catalysis. The anticodon nucleotides involved in base-specific interactions with the enzyme prevail both in the initial binding recognition and in favouring aminoacylation catalysis. Tertiary nucleotides of base pair G19-C56 and base triple U45-G10-C25 contribute primarily to stabilization of the correctly folded tRNA(Phe) structure, which is important for binding. Other nucleotides of the central core (U20, U16 and of the A26-G44 tertiary base pair) are involved in conformational adjustment of the tRNA upon its interaction with the enzyme. The specificity of nucleotide A73, mutation of which slightly reduces the catalytic rate of aminoacylation, is not displayed at the binding step. A few backbone-mediated contacts of PheRS with the acceptor and anticodon stems revealed in the crystal structure do not contribute to tRNA(Phe) discrimination, their role being limited to stabilization of the complex. The highest affinity of T. thermophilus PheRS for cognate tRNA, observed for synthetase-tRNA complexes, results in 100-3000-fold binding discrimination against non-cognate tRNAs.     

Electrical excitability of proteinoid microspheres composed of basic and acidic proteinoids

Material flow equilibration, an endogenous interaction adjustment process underlying fulfillment of material flow continuity during material self-assembly, also underlies electrical excitability observed in the proteinoid microspheres as simulated models of the protocell. The spikings of the membrane potentials are attributed to a singular character of the interaction rate coefficients measuring the strengths of the coupling between basic and acidic proteinoids, in which the rates change singularly with time due to material flow equilibration.     

Proteinoid microspheres and the process of prebiological photophosphorylation

A chemical model of prebiological photophosphorylation with participation of hemoproteinoid microspheres, mixed microspheres containing bonded riboflavin and microspheres obtained from glycine rich proteinoids was studied. The illumination of aqueous solutions containing microspheres, K₂HPO₄, ADP and electron acceptor leads to an increase of ATP concentration and to a decrease of concentration of inorganic phosphate. Initial photochemical reactions with participations of proteinoid microspheres could have evolved in the course of chemical evolution and led to the emergence of the photophosphorylation in its modern biochemical form.     

Protoporphyrin ix as a possible ancient photosensitizer: Spectral and photochemical studies

Due to the potential special position of protoporphyrin IX in the evolution of photosynthesis, the absorption and fluorescence characteristics of this pigment and its complexes with human serum albumin (HSA) and basic proteinoid have been studied in parallel with their photochemical activity. The most significant change in the absorption spectrum of PP IX was the appearance of a new maximum at 455 (or 461) nm in the presence of HSA or proteinoid respectively. Some changes in the physicochemical properties of PP IX in different microenvironments have been detected by changes in fluorescence emission and excitation spectra (intensity, quantum yields, position of maxima). The increase of fluorescence quantum yield resulting from the formation of PP IX complexes with HSA or proteinoid correlates with the increase of their photochemical activity. Results obtained are discussed from the point of view of the early evolution of the photosynthetic apparatus.     

A Potential Yeast Actin Allosteric Conduit Dependent on Hydrophobic Core Residues Val-76 and Trp-79

Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76–79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation.     

DNA substrate specificity of DNA helicase E from calf thymus.

DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair.