Dye-induced aggregation of single stranded RNA: a mechanistic approach

The binding of proflavine (D) to single stranded poly(A) (P) was investigated at pH 7.0 and 25 degrees C using T-jump, stopped-flow and spectrophotometric methods. Equilibrium measurements show that an external complex PD(I) and an internal complex PD(II) form upon reaction between P and D and that their concentrations depend on the polymer/dye concentration ratio (C(P)/C(D)). For C(P)/C(D)<2.5, cooperative formation of stacks external to polymer strands prevails (PD(I)). Equilibria and T-jump experiments, performed at I=0.1M and analyzed according to the Schwarz theory for cooperative binding, provide the values of site size (g=1), equilibrium constant for the nucleation step (K( *)=(1.4+/-0.6)x10(3)M(-1)), equilibrium constant for the growth step (K=(1.2+/-0.6)x10(5)M(-1)), cooperativity parameter (q=85) and rate constants for the growth step (k(r)=1.2x10(7)M(-1)s(-1), k(d)=1.1 x 10(2)s(-1)). Stopped-flow experiments, performed at low ionic strength (I=0.01 M), indicate that aggregation of stacked poly(A) strands do occur provided that C(P)/C(D)<2.5.     

Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.     

Switches, catapults, and chaperones: steady-state kinetic analysis of Hsp70-substrate interactions

Hsp70 chaperones are heterotropic allosteric systems in which ATP and misfolded or aggregated polypeptides are the activating ligands. To gain insight into the mechanism by which ATP and polypeptides regulate Hsp70 chaperone activity, the effect of a short peptide on the K(M) for ATP was analyzed using the Escherichia coli Hsp70 called DnaK. In the absence of peptide, the K(-P)(M) for ATP is 52 +/- 11 nM, whereas this value jumps to 14.6 +/- 1.6 microM in the presence of saturating peptide. This finding supports a mechanism in which ATP binding drives the chaperone in one direction and peptide binding pushes the chaperone back in the opposite direction (and thus increases K(M)), according to ATP + DnaK.P <==> ATP.DnaK.P <==> ATP.DnaK* + P, where ATP.DnaK.P is an intermediate from which competing ATP hydrolysis occurs (ATP.DnaK.P --> ADP.DnaK.P). We show that this branched mechanism can even explain how DnaK hydrolyzes ATP in the absence of peptide and that the true rate constant for DnaK-mediated ATP hydrolysis (k(hy)) in the absence of peptide may be as high as 0.5 s(-)(1) (rather than 5 x 10(-)(4) s(-)(1) as often stated in the literature). What happens is that a conformational equilibrium outcompetes ATP hydrolysis and effectively reduces the concentration of the intermediate by a factor of a thousand, resulting in the following relation: k(cat) = k(hy)/1000 = 5 x 10(-)(4) s(-)(1). How polypeptide substrates and the co-chaperone DnaJ modulate DnaK to achieve its theoretical maximal rate of ATP hydrolysis, which we suggest is 0.5 s(-)(1), is discussed.     

Gap-junctional single-channel permeability for fluorescent tracers in mammalian cell cultures

We have developed a simple dye transfer method that allows quantification of the gap-junction permeability of small cultured cells. Fluorescent dyes (calcein and Lucifer yellow) were perfused into one cell of an isolated cell pair using a patch-type micropipette in the tight-seal whole cell configuration. Dye spreading into the neighboring cells was monitored using a low-light charge-coupled device camera. Permeation rates for calcein and Lucifer yellow were then estimated by fitting the time course of the fluorescence intensities in both cells. For curve fitting, we used a set of model equations derived from a compartment model of dye distribution. The permeation rates were correlated to the total ionic conductance of the gap junction measured immediately after the perfusion experiment. Assuming that dye permeation is through a unit-conductance channel, we were then able to calculate the single-channel permeance for each tracer dye. We have applied this technique to HeLa cells stably transfected with rat-Cx46 and Cx43, and to BICR/M1R(k) cells, a rat mammary tumor cell line that has very high dye coupling through endogenous Cx43 channels. Scatter plots of permeation rates versus junctional conductance did not show a strictly linear correlation of ionic versus dye permeance, as would have been expected for a simple pore. Instead, we found that the data scatter within a wide range of different single-channel permeances. In BICR/M1R(k) cells, the lower limiting single-channel permeance is 2.2 +/- 2.0 x 10(-12) mm3/s and the upper limit is 50 x 10(-12) mm3/s for calcein and 6.8 +/- 2.8 x 10(-12) mm3/s and 150 x 10(-12) mm3/s for Lucifer yellow, respectively. In HeLa-Cx43 transfectants we found 2.0 +/- 2.4 x 10(-12) mm3/s and 95 x 10(-12) mm3/s for calcein and 2.1 +/- 6.8 x 10(-12) mm3/s and 80 x 10(-12) mm3/s for Lucifer yellow, and in HeLa-Cx46 transfectants 1.7 +/- 0.3 x 10(-12) mm3/s and 120 x 10(-12) mm3/s for calcein and 1.3 +/- 1.1 x 10(-12) mm3/s and 34 x 10(-12) mm3/s for Lucifer yellow, respectively. This variability is most likely due to a yet unknown mechanism that differentially regulates single-channel permeability for larger molecules and for small inorganic ions.     

Intrahost human immunodeficiency virus type 1 evolution is related to length of the immunocompetent period.

The antigenic diversity threshold theory predicts that antigenic sites of human immunodeficiency virus type 1, such as the V3 region of the external glycoprotein gp120, evolve more rapidly during the symptom-free period in individuals progressing to AIDS than in those who remain asymptomatic for a long time. To test this hypothesis, genomic RNA sequences were obtained from the sera of 44 individuals at seroconversion and 5 years later. The mean number of nonsynonymous nucleotide substitutions in the V3 region of the viruses circulating in 31 nonprogressors (1.1 x 10(-2) +/- 0.1 x 10(-2) per site per year) was higher than the corresponding value for 13 progressors (0.66 x 10(-2) +/- 0.1 x 10(-2) per site per year) (P < 0.01), while no difference between the mean numbers of synonymous substitutions in the two groups was seen (0.37 x 10(-2) +/- 0.1 x 10(-2) and 0.51 x 10(-2) +/- 0.2 x 10(-2) per site per year for nonprogressors and progressors, respectively; P > 0.1). The mean ratios of synonymous nucleotide p distance to nonsynonymous p distance were 0.35 for nonprogressors and 0.62 for progressors. The number of nonsynonymous substitutions was not associated with virus load or virus phenotype, which are established predictors of disease progression, but correlated strongly with the duration of the immunocompetent period (r2 = 0.41; P = 0.001). This indicates that there is no causative relationship between intrahost evolution and CD4+ cell decline. Our data suggest that intrahost evolution in human immunodeficiency virus type 1 infection is driven by selective forces, the strength of which is related to the duration of the immunocompetent period.     

Pulse radiolysis studies of beta-carotene in oxygenated DMSO solution. Formation of beta-carotene radical cation

The spectroscopic and kinetic characteristics of beta-carotene radical cation (beta-carotene(.+)) were studied by pulse radiolysis in aerated DMSO solution. The buildup of beta-carotene(.+) with k(1) = (4.8 +/- 0.2) x 10(8) dm(3) mol(-1) s(-1) [lambda(max) = 942 nm, epsilon = (1.6 +/- 0.1) x 10(4) dm(3) mol(-1) cm(-1)] results from an electron transfer from beta-carotene to DMSO(.+). The beta-carotene(.+) species decays exclusively by first-order reaction, k = (2.1 +/- 0.1) x 10(3) s(-1), probably by two processes: (1) at low substrate concentration by hydrolysis and (2) at high concentrations also by formation of dimer radical cation (beta-carotene)(2)(.+). Under the experimental conditions, a small additional beta-carotene triplet-state absorption ((3)beta-carotene) in the range of 525 to 660 nm was observed. This triplet absorption is quenched by oxygen (k = 7 x 10(4) s(-1)), resulting in singlet oxygen ((1)O(2)), whose reactions can also lead to additional formation of beta-carotene(.+).     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Folding of a three-stranded coiled coil

Coiled coils consist of two or more amphipathic a-helices wrapped around each other to form a superhelical structure stabilized at the interhelical interface by hydrophobic residues spaced in a repeating 3-4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which association and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three-stranded coiled coil. The designed 29-residue peptide LZ16A was shown previously to be in a concentration-dependent equilibrium between unfolded monomer (M), folded dimer (D), and folded trimer (T). We show by time-resolved fluorescence change experiments that folding of LZ16A to D and T can be described by 2M (k1)<==>(k(-1)) D and M + D (k2)<==>(k(-2)) T. The following rate constants were determined (25 degrees C, pH 7): k1 = 7.8 x 10(4) M(-1) s(-1), k(-1) = 0.015 s(-1), k2 = 6.5 x 10(5) M(-1) s(-1), and k(-2) = 1.1 s(-1). In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far-ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to K(D) = k1/2k(-1) and K(T) = k2/k(-2). Furthermore, pulsed hydrogen-exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are compatible with a two-step reaction in which a subpopulation of association competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.     

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.     

Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.

Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.     

Discriminative power of phrenic twitch-induced dynamic response for diagnosis of sleep apnea during wakefulness.

The diagnosis of the obstructive sleep apnea syndrome relies on polysomnography. Bilateral anterior magnetic phrenic stimulation (BAMPS) mimics the dissociation between upper airway (UA) muscles and diaphragm commands that leads to UA closure during sleep. We evaluated BAMPS as a mean to identify obstructive sleep apnea syndrome patients through the characterization of the UA dynamics in 28 consecutive awake patients (18 apneic and 10 nonapneic). Driving pressure (Pd) and instantaneous flow (V) were recorded in response to BAMPS to determine the point of flow limitation (Vimax) and of minimal flow (Vimin) and the flow-pressure relationship [Vi = (k(1) x Pd) + (k(2) x Pd(2))]. Vimax, Vimin, UA resistance at Vi(min), and the coefficient of the flow-pressure relationship (k(1)) were correlated with apnea-hypopnea index (respectively, R = -0.735, P < 0.0001; R = -0.584, P = 0.001; R = 0.474, P = 0.01; and R = -0.567, P < 0.01). Body mass index was also correlated with apnea-hypopnea index (R = 0.500, P < 0.01). Apneic patients had a lower Vimax (Vimax = 678 +/- 386 vs. 1,247 +/- 271 ml/s; P < 0.001), a lower Vimin (Vimin = 460 +/- 313 vs. 822 +/- 393 ml/s; P < 0.05) and a lower k(1) (k(1) = 162 +/- 67 vs. 272 +/- 112 ml x cmH(2)O x s(-1); P < 0.01) than nonapneic ones. Using a classification and regression tree approach, we found that a Vimax of <803 ml/s (n = 12) selected only apneic patients. When Vimax of >803 ml/s (n = 16), a k(1) of >266.7 ml. cmH(2)O x s(-1) identified only nonapneic patients (n = 5). In 11 cases, Vimax > 803 ml/s and k(1) < 266.7 ml. cmH(2)O x s(-1). These included five nonapneic and six apneic patients. We conclude that UA dynamic properties studied with BAMPS during wakefulness significantly differ between nonapneic and apneic patients.     

Behavior of red king crab larvae: phototaxis, geotaxis and rheotaxis

Zoeae of Paralithodes camtschatica were positively phototactic to white light intensities above 1 x 10(13) q cm-2 s-1. Negative phototaxis occurred at low (1 x 10(12) q cm-2 s-1), but not high intensities (2.2 x 10(16) q cm-2 s-1). Phototactic response was directly related to light intensity. Zoeae also responded to red, green and blue light. Zoeae were negatively geotactic, but geotaxis was dominated by phototaxis. Horizontal swimming speed of stage 1 zoeae < 4 d old was 2.4 +/- 0.1 (SE) cm s-1 and decreased to 1.7 +/- 0.1 cm s-1 in older zoeae (P < 0.01). Horizontal swimming speed of stage 2 zoeae was not significantly different from > or = 4 d old stage 1 zoeae. Vertical swimming speed, 1.6 +/- 0.1 cm s-1, and sinking rate, 0.7 +/- 0.1 cm s-1, did not change with ontogeny. King crab zoeae were positively rheotactic and maintained position in horizontal currents less than 1.4 cm s-1. Starvation reduced swimming and sinking rates and phototactic response.     

Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate

One-electron oxidation of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) by the azide radical generates the radical cation (H(4)B(*)(+)) which rapidly deprotonates at physiological pH to give the neutral trihydrobiopterin radical (H(3)B(*)); pK(a) (H(4)B(*)(+) <==> H(3)B(*) + H(+)) = (5.2 +/- 0.1). In the absence of ascorbate both the H(4)B(*)(+) and H(3)B(*) radicals undergo disproportionation to form quinonoid dihydrobiopterin (qH(2)B) and the parent H(4)B with rate constants k(H(4)B(*)(+) + H(4)B(*)(+)) = 6.5 x 10(3) M(-1) s(-1) and k(H(3)B(*) + H(3)B(*)) = 9.3 x 10(4) M(-1) s(-1), respectively. The H(3)B(*) radical is scavenged by ascorbate (AscH(-)) with an estimated rate constant of k(H(3)B(*) + AscH(-)) similar 1.7 x 10(5) M(-1) s(-1). At physiological pH the pterin rapidly scavenges a range of biological oxidants often associated with cellular oxidative stress and nitric oxide synthase (NOS) dysfunction including hydroxyl ((*)OH), nitrogen dioxide (NO(2)(*)), glutathione thiyl (GS(*)), and carbonate (CO(3)(*-)) radicals. Without exception these radicals react appreciably faster with H(4)B than with AscH(-) with k(*OH + H(4)B) = 8.8 x 10(9) M(-1) s(-1), k(NO(2)(*) + H(4)B) = 9.4 x 10(8) M(-1) s(-1), k(CO(3)(*-) + H(4)B) = 4.6 x 10(9) M(-1) s(-1), and k(GS(*) + H(4)B) = 1.1 x 10(9) M(-1) s(-1), respectively. The glutathione disulfide radical anion (GSSG(*-)) rapidly reduces the pterin to the tetrahydrobiopterin radical anion (H(4)B(*-)) with a rate constant of k(GSSG(*-) + H(4)B) similar 4.5 x 10(8) M(-1) s(-1). The results are discussed in the context of the general antioxidant properties of the pterin and the redox role played by H(4)B in NOS catalysis.     

Co-crystal structure and inhibition of factor Xa by PD0313052 identifies structurally stabilized active site residues of factor Xa and prothrombinase

The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.     

The two modes of binding of Ru(phen)(2)dppz(2+) to DNA: thermodynamic evidence and kinetic studies

The binding of Ru(phen)(2)dppz(2+) (dppz=dipyrido[3,2-a:2',3'-c]phenazine) to DNA was investigated at pH 7.0 and 25 degrees C using stopped-flow and spectrophotometric methods. Equilibrium measurements show that two modes of binding, whose characteristics depend on the polymer to dye ratio (C(P)/C(D)), are operative. The binding mode occurring for values of C(P)/C(D) higher than 3 exhibits positive cooperativity, which is confirmed by kinetic experiments. The reaction parameters are K=2 x 10(3)M(-1), omega=550, n=1, k(r)=(1.9+/-0.5) x 10(7)M(-1)s(-1) and k(d)=(9.5+/-2.5)x10(3)s(-1) at I=0.012 M. The results are discussed in terms of prevailing surface interaction with DNA grooves accompanied by partial intercalation of the dppz residue. The other binding mode becomes operative for C(P)/C(D)<3 and the equilibria analysis shows this is an ordinary intercalation mode (K=1.3 x 10(6) M(-1), n=1.5 at I=0.012 M and K=2 x 10(5) M(-1), n=1.2 at I=0.21 M). Similar behaviour is displayed by double-stranded poly(A).     

A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions.

The leucine zipper is a dimeric coiled-coil protein structure composed of two amphipathic alpha-helices with the hydrophobic surfaces interacting to create the dimer interface. This structure has been found to mediate the dimerization of two abundant classes of DNA binding proteins: the bZIP and bHLH-Zip proteins. Several workers have reported that amino acids in the e and g positions of the coiled coil can modulate dimerization stability and specificity. Using the bZIP protein VBP as a host molecule, we report a thermodynamic scale (delta delta G) for 27 interhelical interactions in 35 proteins between amino acids in the g and the following e positions (g<==>e') of a leucine zipper coiled coil. We have examined the four commonly occurring amino acids in the e and g positions of bZIP proteins, lysine (K), arginine (R), glutamine (Q), glutamic acid (E), as well as the only other remaining charged amino acid aspartic acid (D), and finally alanine (A) as a reference amino acid. These results indicate that E<==>R is the most stable interhelical pair, being 0.35 kcal/mol more stable than E<==>K. A thermodynamic cycle analysis shows that the E<==>R pair is 1.33 kcal/mol more stable than A<==>A with -1.14 kcal/mol of coupling energy (delta delta Gint) coming from the interaction of E with R. The E<==>K coupling energy is only -0.14 kcal/mol. E interacts with more specificity than Q. The R<==>R pair is less stable than the K<==>K by 0.24 kcal/mol. R interacts with more specificity than K. Q forms more stable pairs with the basic amino acids K and R rather than with E. Changing amino acids in the e position to A creates bZIP proteins that form tetramers.     

The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant

The guanidine hydrochloride (GdnHCl) mediated denaturation pathway for the apo form of homodimeric Escherichia coli aspartate aminotransferase (eAATase) (molecular mass = 43.5 kDa/monomer) includes a partially folded monomeric intermediate, M* [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913; Birolo, L., Dal Piaz, F., Pucci, P., and Marino, G. (2002) J. Biol. Chem. 277, 17428-17437]. The present investigation of the urea-mediated denaturation of eAATase finds no evidence for an M* species but uncovers a partially denatured dimeric form, D*, that is unpopulated in GdnHCl. Thus, the unfolding process is a function of the employed denaturant. D* retains less than 50% of the native secondary structure (circular dichroism), conserves significant quaternary and tertiary interactions, and unfolds cooperatively (mD*<==>U = 3.4 +/- 0.3 kcal mol-1 M-1). Therefore, the following equilibria obtain in the denaturation of apo-eAATase: D <==> 2M 2M* <==> 2U in GdnHCl and D <==> D* <==> 2U in urea (D = native dimer, M = folded monomer, and U = unfolded state). The free energy of unfolding of apo-eAATase (D <==> 2U) is 36 +/- 3 kcal mol-1, while that for the D* 2U transition is 24 +/- 2 kcal mol-1, both at 1 M standard state and pH 7.5.     

Kinetic analysis of [35S]dATP alpha S interaction with P2y(1) nucleotide receptor

The kinetics of 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) ([(35)S]dATP alpha S) interaction with membrane fragments of transfected astrocytoma 1321N1 cells, expressing human P2Y(1) receptors, and the same wild-type cells, not expressing P2Y receptors were studied. Binding of this radioligand was observed with both types of membranes, but sites showing slow on-rate were found only on the transfected cells. These "slow" binding sites behaved as a kinetically homogeneous population and their interaction with the radioligand was shown to occur in two steps, R+A(K(A))<==>RA(k(i))<==>(k(-i))(RA), including the relatively slow isomerization of the complex RA into (RA). Evidence was obtained to assign the isomerized ("slow") binding sites on the transfected cells as P2Y(1) receptor sites, differentiated from other binding sites of non-receptor origin by kinetic analysis, and characterised by the kinetic parameters K(A)=59 +/- 19 nM, k(i)=(9.0 +/- 0.8)10(-3)s(-1) and k(-i)=(3.9 +/- 0.7)10(-3)s(-1). [(35)S]dATP alpha S binding, with kinetic criteria, can be of value for differentiation of the receptor sites from non-receptor sites and thus provides solid basis for radioligand assay of P2Y(1) receptors.     

Peptide-protein interactions: photoinduced electron-transfer within the preformed and encounter complexes of a designed metallopeptide and cytochrome c

Photoinduced electron-transfer (ET) occurs between a negatively charged metallopeptide, [Ru(bpy)(2)(phen-am)-Cys-(Glu)(5)-Gly](3-) = RuCE(5)G, and ferricytochrome c = Cyt c. In the presence of Cyt c, the triplet state lifetime of the ruthenium metallopeptide is shortened, and the emission decays via biexponential kinetics, which indicates the existence of two excited-state populations of ruthenium peptides. The faster decay component displays concentration-independent kinetics demonstrating the presence of a preformed peptide-protein complex that undergoes intra-complex electron-transfer. Values of K(b) = (3.5 +/- 0.2) x 10(4) M(-1) and k(obs)(ET)= (2.7 +/- 0.4) x 10(6) s(-1) were observed at ambient temperatures. The magnitude of k(obs)(ET) decreases with increasing solvent viscosity, and the behavior can be fit to the expression k(obs)(ET) proportional to eta(-alpha) to give alpha = 0.59 +/- 0.05. The electron-transfer process occurring in the preformed complex is therefore gated by a rate-limiting configurational change of the complex. The slower decay component displays concentration-dependent kinetics that saturate at high concentrations of Cyt c. Analysis according to rapid equilibrium formation of an encounter complex that undergoes unimolecular electron-transfer yields K(b)' = (2.5 +/- 0.7) x 10(4) M(-1) and k(obs')(ET)= (7 +/- 3) x 10(5) s(-1). The different values of k(obs)(ET) and k(obs')(ET) suggest that the peptide lies farther from the heme when in the encounter complex. The value of k(obs')(ET) is viscosity dependent indicating that the reaction occurring within the encounter complex is also configurationally gated. A value of alpha = 0.98 +/- 0.14 is observed for k(obs')(ET), which suggests that the rate-limiting gating processes in the encounter complex is different from that in the preformed complex.     

Role of the PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity

In addition to its catalytic domain, phosphoinsositide-dependent protein kinase-1 (PDK1) contains a C-terminal pleckstrin homology (PH) domain, which binds the membrane-bound phosphatidylinositol (3,4,5)-triphosphate [PI(3,4,5)P3] second messenger. Here, we report in vitro kinetic, phosphopeptide mapping, and oligomerization studies that address the role of the PH domain in regulating specific autophosphorylation events, which are required for PDK1 catalytic activation. First, 'inactive' unphosphorylated forms of N-terminal His6 tagged full length (His6-PDK1) and catalytic domain constructs [His6-PDK1(Delta PH)] were generated by treatment with Lambda protein phosphatase (lambda PP). Reconstitution of lambda PP-treated His6-PDK1(Delta PH) catalytic activity required activation loop Ser-241 phosphorylation, which occurred only upon trans-addition of 'active' PDK1 with an apparent bimolecular rate constant of (app)k1(S241) = 374+/-29 M(-1) s(-1). In contrast, full length lambda PP-treated His6-PDK1 catalyzed Ser-241 cis-autophosphorylation with an apparent first-order rate constant of (app)k1(S241) = (5.0+/-1.5) x 10(-4) s(-1) but remained 'inactive'. Reconstitution of lambda PP-treated His(6)-PDK1 catalytic activity occurred only when autophosphorylated in the presence of PI(3,4,5)P3 containing vesicles. PI(3,4,5)P3 binding to the PH domain activated apparent first-order Ser-241 autophosphorylation by 20-fold [(app)k1(S241) = (1.1+/-0.1) x 10(-2) s(-1)] and also promoted biphasic Thr-513 trans-autophosphorylation [(app)k2(T513) = (4.9+/-1.1) x 10(2) M(-1) s(-1) and(app)k3(T513) = (1.5+/-0.2) x 10(3) M(-1) s(-1)]. The results of mutagenesis studies suggest that Thr-513 phosphorylation may cause dissociation of autoinhibitory contacts formed between the contiguous regulatory PH and catalytic kinase domains.