Detecting Amyloid-β Aggregation with Fiber-Based Fluorescence Correlation Spectroscopy

Garai, Kanchan; Sureka, Ruchi; Maiti, Sudipta

doi:10.1529/biophysj.106.101485

Cited by 43 publications

(33 citation statements)

References 18 publications

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“…FCS, in different forms, has proved to be an effective tool for following the size of evolving protein aggregates (32)(33)(34). 100 M of both peptides (at 1000:1 unlabeled:tetramethylrhodamine-labeled peptide ratio) were incubated for 6 h under physiological buffer conditions.…”

Section: Resultsmentioning

confidence: 99%

“…ssNMR studies were performed on aggregates of 7S ⌬G25 peptide containing isotopically enriched Val 18 , Phe 19 , Ala 21 , Gly 33 , and Leu 34 residues. These studies revealed that S ⌬G25 fibrils are also rich in ␤-sheet conformations, similar to the amyloid fibrils formed by S and full-length A␤ (20).…”

Section: Resultsmentioning

confidence: 99%

“…Synthesis and Purification of Peptides-The peptides S, S ⌬G25 , S ⌬G29, S G25P , and A␤ ⌬G25 were synthesized, labeled with 5(6)-carboxytetramethyl rhodamine, purified, and characterized by mass spectroscopy as described previously (20,23). The isotopically labeled peptides for ssNMR studies were synthesized by the same procedure but with selective introduction of uniformly 13 33 , and Leu 34 amino acids. Aggregation Kinetics and Solubility-The peptides were dissolved and adjusted to pH 12 with NaOH at different concentrations ranging from 1 to 2 mM and incubated for 30 min.…”

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confidence: 99%

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Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble

Chandrakesan

Bhowmik

Sarkar

et al. 2015

Journal of Biological Chemistry

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A␤ self-assembles into parallel cross-␤ fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of A␤ in its aggregated state. Major biophysical properties of A␤, including this turn, remain unaltered in the central fragment A␤ 18 -35 . Here, we synthesize a single deletion mutant, ⌬G25, with the aim of sterically hindering the hairpin turn in A␤ 18 -35 . We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of A␤ 18 -35 , suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe 19 and Leu 34 residues, observed in full-length A␤ and A␤ 18 -35 , is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel ␤-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel ␤-sheet. We conclude that the self-assembly of A␤ is critically dependent on the hairpin turn and on the contact between the Phe 19 and Leu 34 regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.Alzheimer's disease pathology has been associated with the aggregation of amyloid ␤ (A␤), 6 a 39 -43-amino acid-long peptide (1, 2). In this process, the unstructured monomers of A␤ get converted into amyloid fibrils composed of hairpin-shaped monomeric units assembled in a parallel ␤ sheet arrangement (3, 4). The central part of this hairpin structure consists of a hydrophilic region flanked by hydrophobic ␤-sheet-forming segments at both ends (5-10). This particular shape is believed to be dictated by the specific pattern of hydrophobic and charged residues in the A␤ sequence and has been identified even in soluble A␤ aggregates (7,(11)(12)(13)(14)(15)(16)(17), including the very early stage oligomers of A␤ (18 , and stabilizes the soluble monomeric and oligomeric assemblies of A␤ (19). This hairpin turn thus seems to be a critical factor dictating the self-assembly of A␤ under physiological conditions. Studying the influence of the turn region on the properties of A␤, separately from that of the distal terminal regions and without changing the electrostatics, can yield valuable information on the logic of A␤ assembly.A␤ aggregation appears to be primarily hydrophobic in nature. In fact a contact between hydrophobic regions containing Phe 19 and Leu 34 is one of the earliest contacts formed during the aggregation of amyloid ␤ (18). In a generic hydrophobic aggregate, the burial of the hydrophobic surface can in principle proceed in an ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble

Chandrakesan

Bhowmik

Sarkar

et al. 2015

Journal of Biological Chemistry

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“…4 However, the resolution and the sensitivity were poor due to the low numerical aperture (NA) of the single-mode fiber. While this is still useful for monitoring multiply-labeled objects such as protein aggregates, 5 the device is impractical for single molecule detection. In addition, the instrument still required alignment of the excitation laser into the fiber, and of the fluorescence signal into the small aperture of the typical avalanche photodiode detector.…”

Section: Introductionmentioning

confidence: 99%

Ultracompact alignment-free single molecule fluorescence device with a foldable light path

et al. 2011

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Abstract. Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4×4×18 cm 3 sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

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“…The application of near-field optical probes for FCS measurements on cellular membranes is expected to provide a substantial advantage over the use of arrayed subwavelength apertures since one has the ability to image the cell surface at high spatial resolution [15][16][17], in order to identify areas of interest, prior to conducting FCS measurements on targeted areas of the membrane. Although FCS has been successfully reported using an untapered fiber optic probe [18], the use of fiber-based optical probes with nanometer-sized apertures has only recently been demonstrated in our laboratories [19]. This article reviews our recent advances toward fabrication of near-field optical probes for biological imaging [20] and our initial demonstration of the utility of these probes for FCS measurements in 2D membranes with subdiffraction-limited resolution [19].…”

Section: Introductionmentioning

confidence: 99%

Near-field optical probes provide subdiffraction-limited excitation areas for fluorescence correlation spectroscopy on membranes

Vobornik

Banks

et al. 2009

Pure and Applied Chemistry

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Near-field optical probes have been used to produce a subdiffraction-limited observation area for fluorescence correlation spectroscopy (FCS) experiments on supported membranes. The design of a bent, etched fiber probe that is compatible with biological imaging in an aqueous environment is described. This probe design is used for proof of principle experiments to measure lipid diffusion in a fluid-supported bilayer. A reduction in excitation area of approximately one order of magnitude (relative to a confocal FCS experiment) is obtained with a probe aperture diameter of 140 nm. We also demonstrate a simple approach for modeling the autocorrelation decay due to diffusion within the excitation profile at the near-field scanning optical microscopy (NSOM) probe aperture. The use of probes with smaller apertures is expected to provide an additional order of magnitude reduction in the observation area, thus enabling the study of cellular membranes with higher concentrations of fluorophores than is currently possible with diffraction-limited techniques.

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Detecting Amyloid-β Aggregation with Fiber-Based Fluorescence Correlation Spectroscopy

Cited by 43 publications

References 18 publications

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble

Ultracompact alignment-free single molecule fluorescence device with a foldable light path

Near-field optical probes provide subdiffraction-limited excitation areas for fluorescence correlation spectroscopy on membranes

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