On the mechanism of interaction between histone I and DNA and histone III and DNA

A mechanism is suggested at the molecular level whereby histone I can act as a cross-link, or strut, between two DNA strands involved in packing the DNA molecule into the confined space of the chromosome. The amino acid sequence of the N-terminal region is known (to 72). It is suggested that this portion is composed of three main functionally distinct segments: (1) (amino acids 1–18) that forms a broken a-helix (by pro) packed into the depths of the major groove; (2) (amino acids 19–35), rich in lys, which forms a “roof” over segment (1) with nine ionic bonds to phosphate; and (3) (amino acids 41–69) which forms an a-helix. Thus segments (1) and (2) grip the DNA helix and segment (3) forms a strut between two DNA strands. Presumably the rest of the histone molecule forms a second lys-rich “hand” grasping the second DNA helix.The amino acid sequence of histone III suggests that it provides a variant on this thesis. In this case the ionic links to phosphate and the packing of the major groove are provided by successive segments of the protein (e.g. a sequence -arg-lys- followed by a β-turn or a short segment of a-helix). This covers amino acids 1–87. The next segment (88–114) forms an a-helix and the last segment (115–135) repeats the structure of the first segment. Thus histone III might also form cross-links between two parallel DNA strands but its attachment is markedly asymmetrical with one “hand” composed of 87 amino acids filling over $\text{1}\text{1}\text{2}$ turns of the major groove and the other “hand” composed of only 20 amino acids.     

On the controversy over "fast" and "slow" helix-coil transition rates in polypeptides

The controversy over “fast” and “slow” helix-coil transition rates in polypeptides is discussed. The “slow” results are derived from the assumption that multiple NMR spectra of α-CH and NH groups arise from chemical exchange. In this paper it is shown that such spectra may be obtained without invoking chemical exchange. The multiplicity arises from the difference in helicity of amino acid residues near the ends of the chain by comparison with amino acid residues nearer the middle, and from a polydispersity in molecular weight. As a consequence of this analysis, support is given to the “fast” transition rates.     

Conformations and interactions of excited states. II. Polystyrene, polypeptides, and proteins

Previous fluorescence and phosphorescence studies of aromatic model compounds have been extended to polymers: “atactic” and isotactic polystyrene, seven aromatic poly-(amino acids), and two proteins. We have confirmed previous observations that both forms of polystyrene exhibit strong excimer fluorescence emission at room temperature but not at 77°K. Of the poly(amino acids) (all observed in helix-supporting solvents), poly-L -phenylalanine, poly(α-benzyl-L -aspartic acid), and poly-1-benzyl-L -histidine likewise show excimer emission at room temperature but not at 77°K., while poly-L -tyrosine, poly-L -tryptophan, poly(γ-benzyl-L -glutamic acid), and poly-S-benzyl-L -cysteine exhibit no excimer emission at either temperature. The aromatic residues of bovine serum albumin exhibit only “normal” fluorescence, but, lysozyme appears to be unique among proteins in showing excimer-like tryptophan emission in the native state; its luminescence becomes “normal” upon denaturation. It appears very probable that none of these polymers has a ground-state conformation in which the aromatic groups have face-to-face orientations appropriate for excimer interaction. It is concluded that at room temperature absorption of light can cause local “melting” of regular (usually helical) structures and thus, in some polymers, permit the attainment of a face-to-face arrangement of aromatic rings within the radiative lifetime of their excited singlet states. In certain other polymers (for reasons not clear at present), and in all polymers at 77°K., this does not occur. This concept is extended to provide a bettor basis for understanding the mechanism of formation of the photodimer of thy mine in irradiated DNA.     

Sex difference in distribution and iron responsiveness of the two ferritins of rat cardiac and skeletal muscle

The presence of two electrophoretically and structurally distinguishable forms of ferritin (“fast” and “slow”) in cardiac and skeletal muscle (diaphragm) of the rat was confirmed. Although the total amount of cardiac ferritin showed no difference in concentration in male and female rats, the distribution between the fast and slow species was remarkedly different in the two sexes, the fast form predominating in the cardiac muscle and diaphragm of the female. In agreement with this, the rates of synthesis and of degradation of the fast species were greater in teh female, while the opposite obtained for the male. Iron administration stimulated synthesis of each ferritin species in the cardiac muscle and diaphragm of both sexes. Induction of cardiac connective tissue hypertrophy with isoproterenol inverted the ratio of slow to fast ferritin in female rats, while iron adminsitration along with isoproterenol restored this to normal. It is concluded that the metabolism of ferritin in cardiac and skeletal muscle is sensitive both to sexual status and to iron administration.     

Two pacemaker channels from human heart with profoundly different activation kinetics

Cardiac pacemaking is produced by the slow diastolic depolarization phase of the action potential. The hyperpolarization-activated cation current (If) forms an important part of the pacemaker depolarization and consists of two kinetic components (fast and slow). Recently, three full-length cDNAs encoding hyperpolarization-activated and cyclic nucleotide-gated cation channels (HCN1-3) have been cloned from mouse brain. To elucidate the molecular identity of cardiac pacemaker channels, we screened a human heart cDNA library using a highly conserved neuronal HCN channel segment and identified two cDNAs encoding HCN channels. The hHCN2 cDNA codes for a protein of 889 amino acids. The HCN2 gene is localized on human chromosome 19p13.3 and contains eight exons spanning approximately 27 kb. The second cDNA, designated hHCN4, codes for a protein of 1203 amino acids. Northern blot and PCR analyses showed that both hHCN2 and hHCN4 are expressed in heart ventricle and atrium. When expressed in HEK 293 cells, either cDNA gives rise to hyperpolarization-activated cation currents with the hallmark features of native If. hHCN2 and hHCN4 currents differ profoundly from each other in their activation kinetics, being fast and slow, respectively. We thus conclude that hHCN2 and hHCN4 may underlie the fast and slow component of cardiac If, respectively.     

Mechanisms of the multiphasic kinetics in the folding and unfolding of globular proteins

The multiphasic kinetics of the protein folding and unfolding processes are examined for a “cluster model” with only two thermodynamically stable macroscopic states, native (N) and denatured (D), which are essentially distributions of microscopic states. The simplest kinetic schemes consistent with the model are: N-(fast) → I-(slow) → D for unfolding and N ← (fast)-D₂ ← (slow)-D₁ for refolding. The fast phase during the unfolding process can be visualized as the redistribution of the native population N to I within its free energy valley. Then, this population crosses over the free energy barrier to the denatured state D in the slow phase. Therefore, the macrostate I is a kinetic intermediate which is not stable at equilibrium. For the refolding process, the initial equilibrium distribution of the denatured state D appears to be separated into D₁ and D₂ in the final condition because of the change in position of the free energy barrier. The fast refolding species D₂ is due to the “leak” from the broadly distributed D state, while the rest is the slow refolding species D₁, which must overpass the free energy barrier to reach N. At an early stage of the folding process the amino acid chain is considered to be composed of several locally ordered regions, which we call clusters, connected by random coil chain parts. Thus, the denatured state contains different sizes and distributions of clusters depending on the external condition. A later stage of the folding process is the association of smaller clusters. The native state is expressed by a maximum-size cluster with possible fluctuation sites reflecting this association. A general discussion is given of the correlation between the kinetics and thermodynamics of proteins from the overall shape of the free energy function. The cluster model provides a conceptual link between the folding kinetics and the structural patterns of globular proteins derived from the X-ray crystallographic data.     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at pH 9.0

In the free-living ameba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called “fast,” “intermediate,” and “slow” phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH values. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be either acid phosphatase, or protein tyrosine phosphatase. Based on data of inhibitor analysis, broad substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, and another localization in the ameba cell than of the fast and intermediate phosphatases, it is concluded that only the slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

A rapid two-step purification of rat cystatin C, one major inhibitor of cysteine proteinases

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developed. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.     

Abstracts: Albany 2007, The 15th Conversation

Abstract

Forty nine molecular dynamics simulations of unfolding trajectories of the segment B1 of streptococcal protein G (GB1) provide a direct demonstration of the diversity of unfolding pathway and give a statistically utmost unfolding pathway under the physical property space. Twelve physical properties of the protein were chosen to construct a 12-dimensional property space. Then the 12-dimentional property space was reduced to a 3-dimentional principle component property space. Under the property space, the multiple unfolding trajectories look like “trees”, which have some common characters. The “root of the tree” corresponds to the native state, the “bole” homologizes the partially unfolded conformations, and the “crown” is in correspondence to the unfolded state. These unfolding trajectories can be divided into three types. The first one has the characters of straight “bole” and “crown” corresponding to a fast two-state unfolding pathway of GB1. The second one has the character of “the standstill in the middle tree bole”, which may correspond to a three-state unfolding pathway. The third one has the character of “the circuitous bole” corresponding to a slow two-state unfolding pathway. The fast two-state unfolding pathway is a statistically utmost unfolding pathway or preferred pathway of GB1, which occupies 53% of 49 unfolding trajectories. In the property space all the unfolding trajectories construct a thermal unfolding pathway ensemble of GB1. The unfolding pathway ensemble resembles a funnel that is gradually emanative from the native state ensemble to the unfolded state ensemble. In the property space, the thermal unfolded state distribution looks like electronic cloud in quantum mechanics. The unfolded states of the independent unfolding simulation trajectories have substantial overlaps, indicating that the thermal unfolded states are confined by the physical property values, and the number of protein unfolded state are much less than that was believed before.     

The different electrophoretic forms of post gamma-globulin their antigenic identity and their structural variability.

Post gamma-globulin, first described as a constant component of protein of cerebrospinal fluid and urine from patients with tubular disorders has also been found in other biological fluids. Post gamma-globulin from a single individual always migrated as several bands after storage. Three electrophoretic forms, immunochemically identical, have been isolated by gel chromatography, preparative continuous flow electrophoresis and ion-exchange chromatography. Their molecular weight was found to be approximately between 11 000 to 12 000. No difference between the three forms could be detected. The N-terminal amino acids were found to be Lys, Arg and Leu respectively for the three forms of post gamma-globulin. The "slow" and "fast" forms of post gamma-globulin seemed to differ by elimination of small basic peptides or amino acids from the N-terminal end of the protein. No enzymatic activity of post gamma-globulin was found, but this requires further investigations.     

Analysis of histone messenger RNA of Drosophila melanogaster by two-dimensional gel electrophoresis.

The kinetics of the hydrogen-deuterium exchange reactions of double-helical poly (rI) · poly (rC), single-stranded poly(rC) and poly(rI), inosine, and cytosine- 5′-phosphoric acid have been examined, at various temperatures in the range 20 °C to 52 °C, by stopped-flow ultraviolet spectrophotometry, in the region 270 to 300 nm. For the solution of double-helical poly(rI) · poly(rC), two first-order deuteration reactions were found: a fast one and a slow one. At 25 °C and at pH 7.0, the rate constant was 12.3 s^?1 for the fast reaction, and 0.13 s^?1 for the slow reaction. The rate constant of the fast reaction is nearly equal to that of the single-stranded poly(rC) (12.6 s^?1), and is assigned to the deuteration at the amino hydrogen (that is, free from the C · I hydrogen bond) of the cytosine residue. The slow reaction is attributable to the deuteration of the two hydrogens: the amino hydrogen of rC and imide hydrogen of rI, which are rapidly exchanging with each other within every rC · rI base-pair. From the observed temperature effect on this slow reaction rate, it has been concluded that there are two types of “opening process” that are relevant to the hydrogen exchange reaction; one of them is predominent in the range 47 °C to 52 °C and the other in the temperature region lower than 47 °C. The enthalpy (H) and entropy (S) differences of the “open” and “closed” forms in the former type process are ΔH = 167 kcal per mole and ΔS = 507 e.u., while in the latter ΔH = 8.1 kcal per mole and ΔS = 10 e.u..     

Subunits polypeptide composition of Rubisco in Avena species

Abstract

Rubisco was extracted and purified from leaves of nineteen Avena species. The holoenzyme shows the already known “slow” and “fast” forms after native electrophoresis. The electrophoresis in denaturing conditions revealed a unique polypeptide for the large subunit and two types of polypeptides for the small subunit. The addition of leupeptin, a thiol proteinase inhibitor, during purification of the holoenzyme, reduced microheterogeneity within both subunit types. Subunit composition of the enzyme appears to be a taxonomic tool for distinguishing three species: A. clauda Dur., A. eriantha Dur. and A. canariensis Baum. Rajh. et Baum.     

On the double peak of NMR in the helix—coil transition region

To attempt to resolve the controversy over “fast” and “slow” helix–coil transition rates in polypeptides, nuclear magnetic resonance spectra were measured for monodisperse poly-γ-benzyl-L -glutamate (PBLG). These results were compared with simulated line spectra which were computed by taking the molecular-weight distribution into consideration. Broad but single peaks have been observed in 220 mHz nmr for the α-CH and NH proton resonance spectra in the transition region. The shape of the line changes with the extent of polydispersity. Assuming a fast conversion rate, a molecular model of the helix–coil transition simulates these results. Consequently, the double peak which has been observed in the nmr of polypeptides at the helix–coil transition region is shown to result from the polydispersity in molecular weight.     

A Rapid Two-Step Purification of Rat Cystatin C,One Major Inhibitor of Cysteine Proteinases

ABSTRACT

Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developped. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.     

On the origin of the genetic code

It is argued that three chemical criteria determined the evolution of the genetic code: codon-anticodon pairing; codon-amino acid pairing; amino acid pairing. The first criterium determined the set of interactive nucleotides; the second, the set of nucleotides interactive with amino acids; the third, the set of mutually interactive amino acids. The code resulted from the intersection of these sets. This hypothesis explains the specificity and universality of the code as well as the “choice” of the standard amino acids and nucleotides from among those available in nature. The specific mechanism for codon-amino acid pairing assumed here is the “backwards” (Crick, 1967) Pelc-Welton (1966) models. Three types of evidence support “backwards” pairing: parallel genetic coding of amino acid pairs (Root-Bernstein, 1982); results of binding experiments by Saxinger and Ponnamperuma (1974); reinterpretation of Jungck's (1978) correlations between the properties of amino acids and their respective anticodon nucleotides. The inversion of the code to its present state occurred as a result of the evolution of tRNA molecules which supplanted parallel codon-amino acid interactions with antiparallel codon-anticodon ones. The paper concludes with suggestions for testing the hypothesis.     

抗植物病毒农药“病毒煞”的氨基酸成分分析

本文通过对抗植物病毒农药“病毒煞”的氨基酸成分进行分析,共含有１８种氨基酸,其中脯氨酸、谷氨酸、精氨酸和天冬氨酸含量较高,约占水解氨基酸总量的４７％;含有１９种游离氨基酸,脯氨酸含量最高,占游离氨基酸总量的５１％,说明氨基酸可能是其防病增产的有效成分之一。     

A calculation of the probability of spontaneous biogenesis by information theory.

The Darwin-Oparin-Haldane “warm little pond” scenario for biogenesis is examined by using information theory to calculate the probability that an informational biomolecule of reasonable biochemical specificity, long enough to provide a genome for the “protobiont”, could have appeared in 10⁹ years in the primitive soup. Certain old untenable ideas have served only to confuse the solution of the problem. Negentropy is not a concept because entropy cannot be negative. The role that negentropy has played in previous discussions is replaced by “complexity” as defined in information theory. A satisfactory scenario for spontaneous biogenesis requires the generation of “complexity” not “order”. Previous calculations based on simple combinatorial analysis over estimate the number of sequences by a factor of 10⁵. The number of cytochrome c sequences is about 3·8 × 10⁶¹. The probability of selecting one such sequence at random is about 2·1 ×10^?65. The primitive milieu will contain a racemic mixture of the biological amino acids and also many analogues and non-biological amino acids. Taking into account only the effect of the racemic mixture the longest genome which could be expected with 95 % confidence in 10⁹ years corresponds to only 49 amino acid residues. This is much too short to code a living system so evolution to higher forms could not get started. Geological evidence for the “warm little pond” is missing. It is concluded that belief in currently accepted scenarios of spontaneous biogenesis is based on faith, contrary to conventional wisdom.     

Accumulation of a large component related to thyrotropin subunits in the pituitary of thyroidectomized rats.

Rat pituitary thyrotropin-β subunit immunoreactivity is demonstrated to increase with time after thyroidectomy. This increase is due to free β subunits and a high molecular weight form. The latter is also immunoreactive in α subunit and native thyrotropin assays. Its immunological properties in these three types of assays respectively, suggest that it represents a “bid β subunit” and a “big α subunit” excluded together in Sephadex G-100 chromatography, rather than a “big thyrotropin”. These larger species could be precursor forms of the subunits. This would be in agreement with the concept of separate biosynthesis of the subunits of pituitary glycoproteic hormones.     

Reaction of ribosomal sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid)

The number of sulfhydryl groups in the Escherichia coli ribosome has been measured by titration with 5,5′-dithiobis(2-nitrobenzoic acid). Under denaturing conditions, there are 38.8 ± 1.0 titratable thiols per 70 S ribosome and 22.8 ± 0.3 and 12.9 ± 0.3 titratable thiols per 50 S and 30 S subunits, respectively. Three categories of thiol groups can be distinguished in the native 70 S ribosome, a “fast reacting” class of about 3 residues, a “slow reacting” class of about 10 residues and a “buried” class including about 26 residues. The addition of polyuridylic acid to reaction mixtures protects a fast-reacting thiol in the 30 S subunit belonging to protein S1.The addition of urea to ribosome solutions makes the buried residues titratable. Denaturation occurs as a sharp transition at a urea concentration between 4 and 4.5 m. Urea does not fully dissociate the ribosome into RNA and protein. Instead, in the case of the 30 S subunit, a slowly sedimenting particle forms in the presence of urea, containing roughly 65% of the normal amount of protein.     

Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.