Structural features of the Abeta amyloid fibril elucidated by limited proteolysis.

Although the gross morphology of amyloid fibrils is fairly well understood, very little is known about how the constituent polypeptides fold within the amyloid folding motif. In the experiments reported here, we used trypsin and chymotrypsin to conduct limited proteolysis studies on synthetic amyloid fibrils composed of the Alzheimer's disease peptide Abeta(1-40). In both reactions, the extreme N-terminal proteolytic fragment is released from fibrils as rapidly as it is from the Abeta monomer, while other proteolytic fragments are generated much more slowly. Furthermore, aggregated material isolated by centrifugation of intermediate digestion time points from both proteases contains, in addition to full-length material, peptides that possess mature C-termini but truncated N-termini. These data strongly suggest that the N-terminal region of Abeta is not involved in the beta-sheet network of the amyloid fibril, while the C-terminus is essentially completely engaged in protective-presumably beta-sheet-structure. In both digests, release of the extreme N-terminal fragments of Abeta(1-40) reaches plateau values corresponding to about 80% of the total available Abeta. This suggests that there are two classes of peptides in the fibril: while the majority of Abeta molecules have an exposed N-terminus, about 20% of the peptides have an N-terminus that is protected from proteolysis within the fibril structure. The most likely cause of this heterogeneity is the lateral association of protofilaments into the fibril structure, which would be expected to generate a unique environment for those Abeta N-termini located at protofilament packing interfaces and/or in the interior core region between the packed protofilaments. This suggests that the N-terminal region of Abeta, while not directly involved in the beta-sheet network of the fibril, may contribute to fibril stability by participating in protofilament packing.

Abeta amyloid fibrils possess a core structure highly resistant to hydrogen exchange.

We describe here experiments designed to characterize the secondary structure of amyloid fibrils of the Alzheimer's amyloid plaque peptide Abeta, using hydrogen-deuterium exchange measurements evaluated by mass spectrometry. The results show that approximately 50% of the amide protons of the polypeptide backbone of Abeta(1-40) resist exchange in aqueous, neutral pH buffer even after more than 1, 000 h of incubation at room temperature. We attribute this extensive, strong protection to H-bonding by residues in core regions of beta-sheet structure within the fibril. The backbone amide hydrogens exchange at variable rates, suggesting different degrees of protection within the fibril. These data suggest that it is unlikely that the entire Abeta sequence is involved in H-bonded secondary structure within the amyloid fibril. Future studies using the methods described here should reveal further details of Abeta fibril structure and assembly. These methods also should be amenable to studies of other amyloid fibrils and protein aggregates.

Ultra-high throughput rotary capillary array electrophoresis scanner for fluorescent DNA sequencing and analysis.

We have constructed a rotary confocal fluorescence scanner and capillary array electrophoresis system that is designed to analyze over 1000 DNA sequencing or fragment sizing separations in parallel. Capillaries are arranged around the surface of a cylinder and a rotating objective in the middle of the cylinder excites and collects fluorescence from labeled DNA fragments as they pass the capillary detection window. The capillaries are pressure-filled with a replaceable matrix and the samples are electrokinetically injected in parallel from a stainless steel microtiter plate at the cathode end. We demonstrate that the instrument is capable of producing four-color data from all capillaries at a scan rate of 4 Hz (corresponding to a linear scan velocity of 121 cm/s). M13 sequencing data were obtained using a 128 capillary array mounted in half of the first quadrant of the scanner. In this initial run, read lengths greater than 500 bases were obtained in over 60% of the capillaries.

A three-wavelength labeling approach for DNA sequencing using energy transfer primers and capillary electrophoresis.

Capillary electrophoresis DNA sequencing has been accomplished by using four different energy transfer primers and three fluorescence detection channels. Methods have also been developed to deconvolve the three-color data into the four base concentrations. The nonnegative least squares and model selection method resulted in the best accuracy. The three-color data were compared to sequencing data obtained using four detection channels and four energy transfer primers. The average accuracy rates obtained over three 500 base M13mp18 runs using three-color coding were 96% including 18 uncallable compressions and 99.6% if these compressions are excluded. The average accuracy rate obtained using four-color coding was 96.3% including 18 uncallable compressions and 99.9% if these compressions are excluded. Although it is unlikely that three-color schemes will replace four-color sequencing, these methods have exposed basic concepts that will be useful in the development of higher-order multiplex coding methods for DNA analysis.

DNA sequencing using a four-color confocal fluorescence capillary array scanner.

The design, construction and operation of a four-color capillary array electrophoresis scanner are presented. The use of sensitive energy transfer primers facilitates four-color detection of the DNA sequencing fragments following excitation at a single laser wavelength (488 nm). This scanner collects fluorescence data from up to 25 capillaries in parallel. The resulting four-color image files are automatically reduced to four-color line plots, and a base-calling program (Sax) is used to call the sequence. The performance of this system for DNA sequencing is demonstrated by examining twelve different motifs of the hypervariable region I of human mitochondrial (mt) DNA obtained from a Sierra Leone population.

Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis.

We have designed and synthesized fluorescent oligonucleotide primers having improved fluorescence and electrophoretic properties by exploiting the concept of resonance fluorescence energy transfer (ET). These primers carry a fluorescein derivative at the 5' end as a common fluorescence donor and other fluorescein and rhodamine derivatives attached to a modified thymidine within the primer sequence as acceptors. These primers all have strong absorption at a common excitation wavelength (448 nm) and fluorescence emission maxima of 525, 555, and 605 nm. The fluorescence emission intensity of the ET primers increases as the spacing between the donor and acceptors is increased, and of the spacings studied the strongest fluorescence was observed when the number of nucleotides between the donor and acceptors is 10. The electrophoretic mobilities of the primers were also found to be a function of the spacing between the donor and the acceptors, and mobilities of the single base extension DNA fragments generated with primers (F10F, F10J, F10T, and F10R) is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragments generated with the four ET primers allow four-color DNA sequencing on a capillary electrophoresis DNA sequencer using a single laser line at 488 nm for excitation and without applying mobility shift adjustments. With single-stranded M13mp18 DNA as the template, a typical run with the ET primers on a commercial sequencer provided DNA sequences with 99-100% accuracy in the first 500 bases using 8-fold less DNA template than that typically required using T7 DNA polymerase.

Electroosmotic flow control and surface conductance in capillary zone electrophoresis.

Electroosmotic flow within capillary zone electrophoresis can be altered electronically through a mechanism modeled as surface conductance. Experimentally, a small zone of conductive silver on the outer surface of the capillary is shown to permit good control of electroosmotic flow with an applied external voltage. This control is modeled as capacitance across the capillary wall and conductance along the double layer on the inner surface. Experimental results presented agree with theory developed from this model. Surface conductance in capillary zone electrophoresis may significantly simplify electronic control of electroosmotic flow and lead to a better understanding of the effects of chemical modifications to the inner surface of the capillary.

Effects of buffer pH on electroosmotic flow control by an applied radial voltage for capillary zone electrophoresis.

Electroosmotic flow has been shown to be controlled via an applied radial voltage. Many factors determine the effectiveness of this control, and one major factor is buffer pH. In this study the effectiveness of the applied radial voltage for controlling electroosmotic flow while varying buffer pH is examined. Previously developed theory is applied and compared to experimental results for a pH range from 1.4 to 6.32. Analysis time is dramatically reduced by applying a radial voltage for separation of a peptide mixture at pH 1.4. Theory predicts laminar flow profiles under some conditions when applying this technique. However, experimental evidence at pH 6.32 and 1.4 shows no evidence of band broadening from a laminar flow profile. Theoretical and experimental results indicate the largest range of effective electroosmotic flow control via an applied radial voltage occurs at low ph. Furthermore, a sigmoidal relationship between electroosmotic flow and applied radial voltage is clearly apparent under these conditions. In contrast, at high buffer pH ( > 6) the relationship appears to be linear and is only over a limited range of flow velocities.