Residue-Specific, Real-Time Characterization of Lag-Phase Species and Fibril Growth During Amyloid Formation: A Combined Fluorescence and IR Study of p-Cyanophenylalanine Analogs of Islet Amyloid Polypeptide

Amyloid formation normally exhibits a lag phase followed by a growth phase, which leads to amyloid fibrils. Characterization of the species populated during the lag phase is experimentally challenging, but is critical since the most toxic entities may be pre-fibrillar species. p-Cyanophenylalanine (F_C≡N) fluorescence is used to probe the nature of lag-phase species populated during the formation of amyloid by human islet amyloid polypeptide. The polypeptide contains two phenylalanines at positions 15 and 23 and a single tyrosine located at the C-terminus. Each aromatic residue was separately replaced by F_C≡N. The substitutions do not perturb amyloid formation relative to wild-type islet amyloid polypeptide as detected using thioflavin T fluorescence and electron microscopy. F_C≡N fluorescence is high when the cyano group is hydrogen bonded and low when it is not. It can also be quenched via Förster resonance energy transfer to tyrosine. Fluorescence intensity was monitored in real time and revealed that all three positions remained exposed to solvent during the lag phase but less exposed than unstructured model peptides. The time course of amyloid formation as monitored by thioflavin T fluorescence and F_C≡N fluorescence is virtually identical. Fluorescence quenching experiments confirmed that each residue remains exposed during the lag phase. These results place significant constraints on the nature of intermediates that are populated during the lag phase and indicate that significant sequestering of the aromatic side chains does not occur until β-structure sufficient to bind thioflavin T has developed. Seeding studies and analysis of maximum rates confirm that sequestering of the cyano groups occurs concomitantly with the development of thioflavin T binding capability. Overall, the process of amyloid formation and growth appears to be remarkably homogenous in terms of side-chain ordering. F_C≡N also provides information about fibril structure. Fluorescence emission measurements, infrared measurements, and quenching studies indicate that the aromatic residues are differentially exposed in the fibril state with Phe15 being the most exposed. F_C≡N is readily accommodated into proteins; thus, the approach should be broadly applicable.     

A Human Immunodeficiency Virus Screening Algorithm to Address the High Rate of False-Positive Results in Pregnant Women in Japan

Background

Prenatal human immunodeficiency virus (HIV) testing is essential for the prevention of mother-to-child transmission. However, false-positive results of screening testing are a concern as they may cause unnecessary emotional stress to pregnant women waiting for confirmatory test results. In regions with an extremely low prevalence, the positive predictive values of screening are unacceptably low rate. Here, we propose a HIV screening algorithm consisting of serial two fourth-generation enzyme immunoassays to reduce the number of false-positive screening results.

Methodology/Principal Findings

When 6461 pregnant women presenting to two maternity hospitals located in the Tokyo metropolitan area of Japan from September, 2004 to January, 2006 were tested using Enzygnost HIV Integral as a first screening test, 27 showed positive reactions. When these positive reaction samples were tested using VIDAS HIV DUO Quick as a second screening test, only one of them had a positive reaction, and the remaining 26 were nonreactive. Confirmatory Western blots and nucleic acid amplification test also showed that one was positive and the remaining 26 were negative; the subject who was positive with the confirmatory tests was identical to the subject who was positive with the second screening test. Thus, by adding the second screening test, the false-positive rate was improved from 0.4% to 0%, and the positive predictive value from 3.7% to 100%, compared with the single screening test.

Conclusion

By applying our serial screening algorithm to HIV testing in maternity hospitals, many uninfected pregnant women would not need to receive confirmatory tests and be subjected to emotional turmoil while waiting for their confirmatory test results. This algorithm would be suitable for HIV testing of pregnant women living in low prevalence regions such as Japan.     

Conformational analysis of peptide fragments derived from the peripheral subunit-binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: evidence for nonrandom structure in the unfolded state

There is currently a great deal of interest in the early events in protein folding. Two issues that have generated particular interest are the nature of the unfolded state under native conditions and the role of local interactions in folding. Here, we report the results of a study of a set of peptides derived from a small two-helix protein, the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex. Five peptides of overlapping sequence were prepared, including sequences corresponding to each of the helices and to the region connecting them. The peptides were characterized by CD and, where possible, nmr. A peptide corresponding to the second helix is between 12 and 17% helical at neutral pH. CD also indicates a lower percentage of helical structure in the peptide corresponding to the first alpha-helix, although the values of the alpha-proton chemical shifts suggest some preference for nonrandom structure. Peptides corresponding to the interhelical loop, which in the full domain contains two overlapping beta-turns and a 5-residue 3(10)-helix, are less structured. There is no significant change in the helicity of any of these peptides with pH. To test for fragment complementation, CD spectra of the two peptides derived from each helix and the long connecting peptide were compared to the spectra of each possible pair, as well as to a mixture containing all three. No increase in structure was observed. We complement our peptide studies by characterizing a point mutant, D34V, which disrupts a critical hydrogen bonding network. This mutant is unable to fold and provides a useful model of the denatured state. The mutant is between 9 and 16% helical as judged by CD. The modest amount of helical structure formed in some of the peptide fragments and in the point mutant suggests that the denatured state of the peripheral subunit binding domain is not completely unstructured. This may contribute to the very rapid folding observed for the intact protein.     

Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin

Human chorionic gonadotropin (hCG), is a placental hormone which exerts its major effect by stimulating progesterone production, crucially sustaining the early weeks of pregnancy. Detection of hCG with specific monoclonal antibodies (mAbs) has become the chosen means for pregnancy diagnosis. We have used antibody Fv fragments derived from two high-affinity mAbs, one against the alpha and the other against the beta-hCG subunit to enable the crystallisation of intact or desialylated hCG. Crystals of a ternary complex composed of Fv anti-alpha/hCG/Fv anti-beta were found to diffract to 3.5 A resolution, and the structure was solved by molecular replacement. In the crystal, the two Fvs keep hCG as in a molecular cage, providing good protein-protein contacts and leaving enough space for the saccharides to be accommodated in the cell solvent. The two Fvs were found not to interact directly through their complementary-determining regions with the hCG saccharides, but only with the protein. The hCG structure in the ternary complex was very close to that of the HF partially deglycosylated hormone, thus indicating that neither the saccharides nor the Fvs had any substantial influence on hormone structure.     

Folding of the multidomain ribosomal protein L9: the two domains fold independently with remarkably different rates

The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (Tm = 67 degrees C for the C-terminal domain, 80 degrees C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72 degrees C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69 degrees C were used to determine the folding rate constant, 3.3 s-1, and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1, respectively, for the N-terminal domain at 25 degrees C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N- and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.     

Rapid cooperative two-state folding of a miniature alpha-beta protein and design of a thermostable variant

There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed alpha-beta protein (NTL9(1-39)) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20mM sodium acetate, 100mM NaCl the temperature of maximum stability is 6 degrees C, the t(m) is 65.3 degrees C, deltaH degrees (t(m)) is between 24.6 kcalmol(-1) and 26.3 kcalmol(-1), and deltaC(p) degrees is 0.38 kcalmol(-1)deg(-1). The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/2H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640 s(-1) and the value of deltaG degrees calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t(m) of 82 degrees C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state.     

Female Response to Reduced Male Parental Care in Birds: An Experiment in Tree Swallows

In biparental species, parental-investment theory generally predicts an inverse relationship between the level of parental care provided by each parent and incomplete compensation by one parent in response to reduced parental care by their partner. The factors that influence the magnitude of this compensation have rarely been examined in birds. For example, the level of compensation may differ between a widowed bird that receives no assistance from its partner and a mated bird whose partner is still present but providing less than its normal share of parental care. This study compares the compensatory response of female tree swallows (Tachycineta bicolor) without their mate's parental assistance (when females are widowed) and with reduced male parental care (when males are handicapped by cutting some feathers). When compared with control females, experimental females compensated more in terms of nest visits for the absence than the reduction of male parental care. In addition, widowed females had significantly reduced brood mass and fledging success compared with control females. Although handicapped males reduced their nest-visit rate significantly, females with handicapped mates did not significantly increase their nest-visit rate nor was there reduced brood mass or reduced fledging success at their nests. Total nest-visit rate was similar for all groups, yet widowed females fledged fewer and lighter young, suggesting that they brought less food per nest visit. We suggest that fledging success and measures of offspring quality are probably better indicators of the level of compensatory parental care than nest-visit rate. We suggest that for widowed females the benefits of a relatively large compensatory response outweighed the costs; whereas, for females with a handicapped mate the benefits of higher feeding rates were not greater than the cost. The results of this study help to explain the differences among experimental studies of compensatory parental care and point to a new method of testing models of parental care.     

Effects of breeding density, synchrony, and experience on extrapair paternity in tree swallows

Breeding density, synchrony, and experience are expected toinfluence the frequency of extrapair paternity in birds. UsingDNA fingerprinting, we examined the effect of these factorson tree swallows nesting at relatively high (grids of nest-boxes)and low (solitary boxes at least 100 m from the nearest neighbor)densities and in relatively synchronous (Alberta) and asynchronous(Ontario) populations in Canada. The mean percentage of extrapairoffspring per nest did not differ significantly between birdsnesting in grids (43%, n = 22 families) and solitary boxes (57%,n = 12 families). Similarly, there was no significant differencein the mean percentage of extrapair offspring per nest betweenrelatively synchronous (60%, n = 12 Alberta families) and asynchronous(41%, n = 22 Ontario families) populations. We also found noconsistent pattern between extrapair paternity and breedingexperience among seven males and seven females examined overtwo to three breeding seasons. Female tree swallows can influencethe fertilization success of extrapair males by active selectionand rejection of copulation partners. We suggest that this abilitylimits the predicted effect of various ecological factors onthe frequency of extrapair paternity     

Reactivity of chemically generated superoxide radical anion with peroxides as determined by competition kinetics

A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. High pressure liquid chromatography is used as an assay system for $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene.