Equilibrium studies of zinc(II) and cobalt(II) binding to tripeptide analogues of the amino terminus of human serum albumin

Serum albumin is known to bind several divalent metal ions at the amino terminus of the protein. Two peptide analogues for the amino terminus of human albumin, L-aspartyl-L-alanyl-L-histidine-N-Methyl amide (AAHNMA) and glycylglycyl-L-histidine-N-methyl amide (GGHNMA) have been synthesized, and their interactions with Zn(II) and Co(II) ions have been studied using analytical potentiometry. The stability constants of the species and their distribution as a function of pH were determined in 0.16-M KNO₃ at 25°. Comparison of the modes of interaction of the Zn(II) and Co(II) with each of the above peptides indicate that, although Co(II) is a valuable tool for the study of Zn(II) interaction with metalloenzymes, it is considerably less useful as a Zn(II) model with small peptide molecules. The potentiometric properties of the two peptide-Zn(II) systems have been compared to the potentiostatic properties of the albumin-Zn(II) system. The results indicate that AAHNMA is a better analogue for the Zn(II)-HSA interaction than is GGHNMA. The findings suggest that the Zn(II)-HSA binding site is best described as a compound site containing both a histidyl and a neighboring carboxyl group.     

On the problem of comparing protein structures: Development and applications of a new method for the assessment of structural similarities of polypeptide conformations

The development of prediction schemes and the search for evolutionary relationships amongst proteins require reliable methods for the comparison of polypeptide structures. It is shown that methods which attempt to describe structural similarities by a single value generally do not yield reliable estimates of the relatedness of two conformations. A new method is reported, called the D_k procedure, which yields a spectrum of deviations between two structures. Each particular D_k value is a measure of the similarity of the diagonals of the distance matrices of the compared conformations, k being the distance of the diagonals relative to the main diagonal.The method has the following features. (1) D_k is independent of chain length; (2) the method yields the relatedness of two conformations in terms of different structural levels; (3) D_k is a high-speed algorithm; (4) the D_k deviations of random structures from any particular conformation are predictable.The following applications are reported. (1) The success of both secondary and tertiary structure predictions are measured in terms of a reliable quality index. (2) The route of a conformation during simulation studies is followed on different structural levels, which exhibit the characteristics of the simulation. (3) The significance of hypotheses on protein folding subject to prediction schemes can be established. (4) A priori information (fixing pieces of secondary structure derived from X-ray investigations during prediction) is extractable from the predicted structures. (5) The evolutionary relatedness of two nucleotide binding proteins is established.The simplicity and speed of the D_k procedure allow its implementation even on minicomputers.     

Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme

Proton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called “occluded” intermediary state of the reaction cycle of the intact enzyme. Despite a K_d in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP⁺ is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2′-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD⁺ by NADH, indicating that 3-acetylpyridine-NAD⁺ and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2′-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424-Arg425-Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent.     

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp²⁴¹ is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310-395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.     

Kinetics of denaturation of DNA

The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix–coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix–coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single-strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.     

Crystal structure of an inhibitor and a model for inhibition of replicative DNA synthesis

6-(p-Hydroxyphenylhydrazino)-uracil is an antimicrobial agent that selectively blocks replicative DNA synthesis in Bacillus subtilis by inhibiting DNA polymerase III. The drug crystallizes as a monoclinic monohydrate, space group $\text{C2}\text{c}$, with $\text{a = 23.920(6)}\text{A}\text{̊}\text{, b = 5.587(3)}\text{A}\text{̊}\text{, c = 17.466(5)}\text{A}\text{̊}\text{, β = 101.45(8) °}$, and eight hydrated molecules per cell. Three-dimensional X-ray diffraction data were collected. The structure was solved by Patterson methods and refined to an R value of 6.8% for the 1651 data. The geometry of the uracil ring is unusual. The bond distances suggest that a resonance form involving a positively charged hydrazino nitrogen and a negatively charged carbonyl oxygen, O(4), makes a large contribution to the valence bond structure of this compound. The exocyclic C(6)N bond is short (1.335 Å), the C(6)C(5) bond distance is 1.371 Å, which is longer than in uracil, and the C(5)C(4) distance (1.396 Å) is short. The uracil ring, the linked hydrazino nitrogen, and the hydrogen on this nitrogen are in the same plane. Each uracil group is hydrogen bonded to a nearly coplanar uracil across a center of symmetry. The water molecule is also near the plane of these paired bases and forms a hydrogen bond with the uracil-linked hydrazino NH group. This paired base arrangement and the restricted rotation about the exocyclic C(6)N link that constrains the hydrazino NH group to lie near the uracil plane suggest a model for the interaction of the drug with template-primer DNA. The drug acts when cytosine is the base to be copied in the template strand, and the drug is competitive with dGTP. Both cytosine and guanine can be accommodated with little distortion of the crystal structure geometry in a manner compatible with the known geometry of DNA. The structural and biochemical aspects of the model for drug action are discussed.     

Estimating the total number of nucleotide substitutions since the common ancestor of a pair of homologous genes: Comparison of several methods and three beta hemoglobin messenger RNA's

Summary The mRNA sequences of beta hemoglobin for human, mouse and rabbit were examined. Observations included the following: (1) there is a significant bias against the use of codons only one nucleotide different from terminating codons; (2) less than 4% of the codons end in adenine; (3), guanine is the most common third position nucleotide but it never follows a second position cytosine; (4) nearest neighbor (doublet) nucleotides are non-random with the greatest contributor to non-randomness being the third position suggesting that codon choice for a given amino acid rather than a choice among amino acids is the more important contributor; (5) the CG dinucleotide is even rarer in positions other than the first and second of the codon than it is in those two, suggesting that the need for arginine has in fact elevated the CG frequency in those positions; (6) 77 per cent of the nucleotides are unsubstituted among these three taxa, which could be a sampling effect, but there is strong evidence that about one-third of them are in fact unsubstitutable because of selective constrainsts; (7) the two longest stretches of unsubstituted nucleotides (32 and 35 consecutive nucleotides) surround the points of the two non-coding insertion sequences; (8) over half the substitutions occur in the third nucleotide position of the codons; (9) silent (non-amino acid changing) substitutions occur at about four times the rate of non-silent substitutions on the basis of their relative opportunity to occur; (10) silent substitutions occur slightly but significantly more often in codons that also have non-silent substitutions than independence of the two events would predict; (11) substitutions occur in adjacent nucleotides significantly more often than chance would predict; (12) among four-fold degenerate codons, third position transitions (principally cytosine-uracil interchanges) outnumber transversions by two to one although the reverse ratio would be expected.The analysis of these messengers provided an opportunity to evaluate the random evolutionary hit (REH) theory. I observed that: (1) the REH theory is premised upon five assumptions, all false; (2) the theory leads to contradictory estimates of the number of varions; (3) the REH values are underestimates; (4) the REH values frequently violate the triangle inequality; (5) the REH values, contrary to claim, are not concordant either with accepted point mutations (PAMs) or augmented distances; (6) the REH values are more likely than values uncorrected for multiple substitutions to give incorrect phylogenies; and (7) the REH values have statistical problems probably associated with a large variance in its fundamental parameter, r_e. From this I conclude that REH theory is not suitable for its intended purpose of estimating from protein sequences of nucleotide substitutions since the common ancestor of two gene products.     

The Suppressor of Killer of prune, a unique glutathione S-transferase

The prune-Killer of prune conditional dominant, lethal interaction in Drosophila was identified in the 1950s, but its mechanism remains unknown. We undertook a genetic screen for suppressors of this lethal interaction and identified a gene we named, Suppressor of Killer of prune Su(Kpn). Su(Kpn) is a unique protein with four N-terminal FLYWCH zinc-finger domains, an acidic domain and a C-terminal glutathione S-transferase (GST) domain. The GST domain of Su(Kpn) is of particular interest because GSTs are usually independent of other protein domains. While GSTs are generally thought of as detoxifying enzymes, they are also associated with cellular toxicity. We predict that the GST domain of the Su(Kpn) creates a toxic product in prune-Killer of prune flies that is lethal. The substrate of the Su(Kpn) remains unknown.     

Segregation of a population in an environment

A mathematical model for spatial patterns and the segregation of a population is presented. Individuals in a population are assumed to move at random under the influence of a given environment potential V(x). The notion of kinetic excitation K(x) and intensity excitation Q(x) of a population is introduced. Then equilibrium states of a population are defined through a macroscopic relation K(x) + Q(x) + V(x) = constant. The problem of finding out equilibrium distributions is reduced to an eigenvalue problem. It is shown that a population is segregated by the nodal surfaces of the eigenfunctions, if it is excited. Some applications of the model to biological and ecological problems are indicated.     

Some relations between different characteristics of selection

Consider the action of selection with fitness w(x) on a quantitative trait x. What selection, among those that produce the same value of selection differential, leads to minimal values of (a) genetic load, (b) variance of the relative fitness, and (c) variance of the trait after selection? We have shown that for (a) and (c) the answer is strict truncation, whereas for (b) the answer is linear selection. The results for (a) and (b) are true for any selection, while the result for (c) is true only for directional selection. Implications of these findings are discussed.     

A model of synchronization of motor acts to a stimulus sequence

A closed-loop timing model is proposed that accounts for several phenomena observed in tasks which require production of a sequence of motor acts in synchrony with a sequence of stimuli. In contrast to the previous models, variables available to the central nervous system of a subject (internal variables) and externally measurable variables are distinguished, and several physiologically justifiable internal variables are included. The model assumes the existence of (a) an internal time-keeper producing a reference interval that is used in a motor-control unit for timing of the next motor command; (b) an intrinsic (subjective) synchrony that relies on some a posteriori (feedback) information about the already executed onset of the motor act. A two-way error-corrective mechanism is hypothesized: (1) period (inverted frequency) corrections — the reference interval (period) is set at the beginning of the task according to the interstimulus-onset interval (s) and later corrected for differences between its duration and the actual duration of s; (2) phase corrections — internal synchronization errors (i.e., time gaps between the central temporal availability of internal representations of stimuli and of some feedback aspect of responses) are corrected for directly in the motor-control unit. Objectively measured systematic asynchrony of responses and stimuli is determined by the internal delays in information transduction. Finally, the model is used for making predictions of a subject's performance in some other experimental settings of the synchronization task.The core of this study was presented at the 4th Workshop on Rhythm Perception and Production, June 1992, Bourges, France (Mates 1992)     

Affinity labeling of stellacyanin by Cr(II) aquo ions

Stellacyanin, the single blue copper protein from $\text{Rhus}$$\text{vernicifera}$, is reduced stoichiometrically by Cr(II)_aq ions yielding a 1:1 adduct between the Cr(III) produced and the reduced protein. This Cr(III)-labeled stellacyanin is substitution inert and no significant loss of the label occurs during extensive dialysis for more than a week. Oxidation by O₂ of the Cr(III)-labeled Cu(I) stellacyanin does not cause the loss of Cr(III) either. Furthermore, reduction of the Cr(III)-labeled stellacyanin Cu(II) by a second equivalent of Cr(II) may be attained without any further labeling. Thus, the one mole of Cr ions binds to stellacyanin during the first reduction step and is most probably coordinated at a specific locus on that protein.     

Evidence of membrane transformation during melanogenesis. Electron microscopic study on the retinal pigment epithelium of chick embryos

Summary Some characteristics of early premelanosomes (PM) suggest that primarily a continuous cisternal complex of the endoplasmic reticulum (ER) is transformed simultaneously to PM. These characteristics are: (i) the form and size, which are similar to ER cisternae; (ii) the localization in groups in the ER; (iii) the same stage of maturation within a group; (iv) the continuities between early PM, and (v) the lack of continuities between ER and PM. Comparative measurements reveal that the limiting membrane of PM, with a total thickness of 7.6±0.19 nm and a center-to-center distance of 5.2±0.06 nm, is significantly thicker than the ER membrane (6.3±0.15 nm and 4.3±0.04 nm, respectively) and the melanosome limiting membrane (6.5±0.22nm and 4.4±0.05 nm, respectively). Therefore, during the formation of melanosomes, the limiting membrane must be transformed from a thin (ER) to a thick (PM) and again to a thin (melanosome) state.     

The development of a novel vector for construction of a full-length cDNA library

A new original vector pEM-(dT)₄₀(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The vector pGEM-(dT)₄₀f(+) is initially transformed into single stranded and then into a linear form and its (dT)₄₀ tail at the 3′-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector circularization and synthesis of the second strand cDNA. This approach has the following advantages: (1) it significantly simplifies cDNA library construction, which includes three steps; (2) full-length cDNA library construction is achieved by adding a (dC)_n homopolymer tail to the 5′end; (3) preparation of a clone library requires a few milligrams of total RNA; (4) it is possible to obtain cDNA clones up to 10 kbp; (5) it does not require PCR reaction (which can induce artifact mutations in cDNA sequences); (6) this approach does not employ restrictase treatment and chimeric cDNA products are not formed.     

Effects of catechol ring fluorination on cardiovascular and renal activities of fenoldopam enantiomers

SK&F 87516 is a potent DA₁ receptor agonist with demonstrated renal vasodilator activity. SK&F 87516 is the 6-fluoro analog of another DA₁ agonist/renal vasodilator agent, fenoldopam. SK&F 87516 is a racemic mixture of two enantiomers, SK&F(R)-87516 and SK&F(S)-87516, and like fenoldopam, the (R)-enantiomer is responsible for the biological activities of the racemate. SK&F(R)-87516 is diuretic in spontaneously hypertensive rats and in dogs, whereas its enantiomer, SK&F(S)-87516 is inactive. SK&F(R)-87516 increases glomerular filtration rate, an effect which may account, in part, for its diuretic activity. Unlike fenoldopam, SK&F(R)-87516 is not associated with acute hypotensive activity, tachycardia, or stimulation of the renin-angiotensin-aldosterone system. The activity differences between SK&F(R)-87516 and fenoldopam are not related to differences in DA₁ agonist potency. The activity differences may be due to the differing effects of fluorine and chlorine on the electron distribution in the catechol ring, resulting in an enhanced effect of SK&F(R)-87516 at α₂-adrenoceptors. © 1994 Wiley-Liss, Inc.     

A Co(III) derivative of concanavalin A

Co(III) has been stoichiometrically incorporated into jack bean concanavalin A. The Co(III) protein still possesses a binding site for an additional divalent transition metal ion which together with Ca(II) can induce the sugar binding ability. No H₂O₂ oxidation of Co(II) occurs with demetallized concanavalin A activated with Ca(II) and Co(II) unless Co(II) is present in a stoichiometric excess. Evidence is presented to indicate that kinetically stable Co(III) is bound to a completely different location than the thermodynamically stable Co(II) protein site.     

Blue-light-induced phosphorylation of a plasma-membrane protein from phototropically sensitive tips of maize coleoptiles

Tips of maize coleoptiles, which function as esential light sensors for the phototropic growth reaction, exhibit a rapid blue-light-induced phosphorylation of a plasma-membrane-associated 100-kDa protein. Characteristics of this reaction are as follows: (i) The functional unit involved in the light-dependent phosphorylation consists of a photoreceptor, a protein kinase and the 100-kDa protein. This complex is only localized in the plasma membrane of tips but not in other parts of the seedling, (ii) The photoreceptor is a cryptochrome-like compound, (iii) The pH optimum of the light-dependent phosphorylation on isolated plasma membranes is around pH 7.8 whereas the light-independent phosphorylation of other membrane proteins occurs at lower values (pH 6.2). (iv) The light-induced in-vitro phosphorylation of the 100-kDa protein is strongly inhibited by the protein-kinase inhibitor staurosporine (IC₅₀=4 nM). (v) The ³²P-moiety of a ³²P-[100 kDa]-protein complex generated after a light pulse with the aid of a membrane-associated protein kinase in the presence of [γ-³²P]ATP cannot be removed by a 100-fold higher level of (unlabelled) ATP. This fact indicates that protein and phosphate are covalently connected and that the complex is not a short-lived intermediate. (vi) The 100-kDa protein is not identical to the plasma-membrane H⁺-ATPase, as shown by immunostaining on Western blots. (vii) Irradiation-dependent in vivo phosphorylation of the 100-kDa protein in tips is already saturated by a light pulse of 5 s. In contrast, the de-phosphorylation of the protein in the dark is a slow reaction lasting about 30 min. It is suggested that the blue-light-triggered phosphorylated status of the 100-kDa protein is an early step in phototropism of the coleoptile, affecting the transport of auxin primarily in the irradiated flank.     

NMR experiments reveal distinct antibody-bound conformations of a synthetic disaccharide representing a general structural element of bacterial lipopolysaccharide epitopes.

The recognition reactions between a synthetic disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and two monoclonal antibodies (mAbs) were studied by NMR, yielding two distinct bound conformations of the carbohydrate ligand. One mAb, S23-24, recognizes the disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with an approximately 10-fold higher affinity than to the disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl. Compared to S25-2, S23-24 binds to alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl with an approximately 50-fold increased affinity. We used NMR experiments that are based on the transferred NOE effect, specifically, trNOESY, trROESY, QUIET-trNOESY, and MINSY experiments, to show that the (2-->8)-specific mAb, S25-2, stabilizes a conformation of the alpha-(2-->4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl, one that is highly populated in aqueous solution and another conformation that is similar to the one bound by S25-2. This is the first example where it is experimentally shown that a carbohydrate ligand may adopt different bioactive conformations upon interaction with mAbs with different fine specificities. Our NMR studies indicate that a careful examination of spin diffusion is critical for the analysis of bioactive conformations of carbohydrate ligands.