Insights from a Convocation: Integrating Discovery-Based Research into the Undergraduate Curriculum.

The National Academies of Sciences, Engineering, and Medicine organized a convocation in 2015 to explore and elucidate opportunities, barriers, and realities of course-based undergraduate research experiences, known as CUREs, as a potentially integral component of undergraduate science, technology, engineering, and mathematics education. This paper summarizes the convocation and resulting report.

Amyloid fiber formation in human γD-Crystallin induced by UV-B photodamage.

γD-Crystallin is an abundant structural protein of the lens that is found in native and modified forms in cataractous aggregates. We establish that UV-B irradiation of γD-Crystallin leads to structurally specific modifications and precipitation via two mechanisms: amorphous aggregates and amyloid fibers. UV-B radiation causes cleavage of the backbone, in large measure near the interdomain interface, where side chain oxidations are also concentrated.

Structural and sequence analysis of the human γD-crystallin amyloid fibril core using 2D IR spectroscopy, segmental 13C labeling, and mass spectrometry.

Identifying the sequence and structural content of residues that compose the core of amyloid fibrils is important because core regions likely control the process of fibril extension and provide potential drug targets. Human γD-crystallin is an eye lens protein that aggregates into amyloid fibrils under acidic conditions. In this manuscript, we use a pepsin enzymatic digest to isolate the core of the amyloid fibrils.

Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils.

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly (13)C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets.

Differential effects of Phe19 and Phe20 on fibril formation by amyloidogenic peptide A beta 16-22 (Ac-KLVFFAE-NH2).

The sequence KLVFFAE (A beta 16-22) in Alzheimer's beta-amyloid is thought to be a core beta-structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in beta-sheet. To elucidate the mechanism of the initial folding process, we undertook combined X-ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized A beta 16-22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild-type (WT) A beta 16-22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab-like beta-crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction.

Self-aggregation of a polyalanine octamer promoted by its C-terminal tyrosine and probed by a strongly enhanced vibrational circular dichroism signal.

The eight-residue alanine oligopeptide Ac-A(4)KA(2)Y-NH(2) (AKY8) was found to form amyloid-like fibrils upon incubation at room temperature in acidified aqueous solution at peptide concentrations >10 mM. The fibril solution exhibits an enhanced vibrational circular dichroism (VCD) couplet in the amide I' band region that is nearly 2 orders of magnitude larger than typical polypeptide/protein signals in this region. The UV-CD spectrum of the fibril solution shows CD in the region associated with the tyrosine side chain absorption.

Different modes of binding of mono-, di-, and trihalogenated phenols to the hemoglobin dehaloperoxidase from Amphitrite ornata.

The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP.

Fiber diffraction as a screen for amyloid inhibitors.

Targeting the initial formation of amyloid assemblies is a preferred approach to therapeutic intervention in amyloidoses, which include such diseases as Alzheimer's, Parkinson's, Huntington's, etc., as the early-stage, oligomers that form before the development of beta-conformation-rich fibers are thought to be toxic. X-ray patterns from amyloid assemblies always show two common intensity maxima: one at 4.

Formation of amyloid fibrils in vitro by human gammaD-crystallin and its isolated domains.

Purpose: Amyloid fibrils are associated with a variety of human protein misfolding and protein deposition diseases. Previous studies have shown that bovine crystallins form amyloid fibers under denaturing conditions and amyloid fibers accumulate in the lens of mice carrying mutations in crystallin genes. Within differentiating lens fiber cells, crystallins may be exposed to low pH lysosome compartments.

Elucidation of residue-level structure and dynamics of polypeptides via isotope-edited infrared spectroscopy.

Infrared spectroscopy is a powerful tool for analyzing the structure of proteins and peptides. The amide I band is particularly sensitive to the strength and position of the hydrogen bonds that define secondary structure as well as dipole-dipole interactions that are affected by the geometry of the peptide backbone. The introduction of a single (13)C-labeled carbonyl into a peptide backbone results in a resolvable shoulder to the main amide I band, which can be analyzed as a separate peak.

The alpha-helix folds more rapidly at the C-terminus than at the N-terminus.

The helix-coil dynamics of different sections of an alpha-helical model peptide were observed separately by nanosecond temperature jump experiments with IR detection on a series of isotopically labeled peptides. The results show that the helix-coil dynamics of the alpha-helical C-terminus are faster than those of the N-terminus.

Experimental evidence for the reorganization of beta-strands within aggregates of the Abeta(16-22) peptide.

Amyloidogenic deposits that accumulate in brain tissue with the progression of Alzheimer's disease contain large amounts of the amyloid beta-peptide. A small fragment of this peptide, comprising residues 16-22 (Abeta(16-22)), forms beta-sheets in isolation, which then aggregate into amyloid fibrils. Here, using isotope edited infrared spectroscopy to probe the secondary structure of the peptide with residue level specificity, we are able to show conclusively that the beta-sheets formed are antiparallel and, following an anneal cycle or prolonged incubation, are in register with the central residue (Phe19) in alignment across all strands.

Intersheet rearrangement of polypeptides during nucleation of {beta}-sheet aggregates.

Many neurodegenerative diseases are characterized by the accumulation of amyloid fibers in the brain, which can occur when a protein misfolds into an extended beta-sheet conformation. The nucleation of these beta-sheet aggregates is of particular interest, not only because it is the rate-determining step toward fiber formation but also because early, soluble aggregate species may be the cytotoxic entities in many diseases. In the case of the prion peptide H1 (residues 109-122 of the prion protein) stable amyloid fibers form only after the beta-strands of the peptide have adopted their equilibrium antiparallel beta-sheet configuration with residue 117 in register across all strands.

Correlations among morphology, beta-sheet stability, and molecular structure in prion peptide aggregates.

The misfolding of proteins into beta-sheets and the subsequent aggregation of these sheets into fibrous networks underlies many diseases. In this paper, the role of peptide structure in determining the ordering of beta-sheet aggregates and the morphology of fibrils and protofibrils is dissected. Using a series of peptides based on residues 109-122 of the Syrian hamster prion protein (H1) with a range of substitutions at position 117, the link between side chain interactions and beta-sheet thermal stability has been investigated.

Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study.

2,2,2-Trifluoroethanol (TFE) is widely used to induce helix formation in peptides and proteins, but the mechanism behind this effect is still poorly understood. Several recent papers have proposed that TFE acts by selectively desolvating the peptide backbone groups of the helix state. Infrared (IR) spectroscopy of the amide I band of polypeptides can be used to probe both secondary structure and backbone solvation, making this technique well suited for addressing the effect of TFE on polypeptide conformation.

Nature of vibrational coupling in helical peptides: an isotopic labeling study.

Infrared (IR) and vibrational circular dichroism (VCD) spectra were measured for a series of isotopically ((13)C on two or more amide Cdouble bond]O) labeled, 25 residue, alpha-helical peptides of the sequence Ac-(AAAAK)(4)AAAAY-NH(2) that were also studied in the previous paper. Theoretical IR and VCD simulations were performed for correspondingly isotopically labeled Ac-A(24)-NHCH(3) constrained to an alpha-helical conformation by use of property tensor transfer from density functional theory (DFT) calculations on Ac-A(10)-NHCH(3). The simulations predicted and experiments confirmed that the vibrational coupling constants between i, i + 1 and i, i + 2 residues differ in sign, thus leading to a reversal of the (13)C VCD pattern and explaining the large shift in the (13)C amide I frequency as reported in the previous paper.

Empirical relationships between isotope-edited IR spectra and helix geometry in model peptides.

Infrared spectroscopy (IR) is commonly used to study secondary structure of both peptides and proteins. The amide I band is very sensitive to peptide secondary structure, and the conformation of a peptide can be probed at the residue level by introducing site-specific isotope-labels into the peptide backbone. The replacement of a carbonyl (12)C with a (13)C results in a approximately 40 cm(-1) shift in the amide I' band.

The organization and assembly of a beta-sheet formed by a prion peptide in solution: an isotope-edited FTIR study.

Insight into the details of protein misfolding diseases requires a detailed understanding of the conformation and dynamics of multistrand beta-sheet aggregates. Here, we report an isotope-edited FTIR study of a model peptide directed at the elucidation of residue-level details of the structure and mechanism of a beta-sheet aggregate. A series of specifically isotope-labeled derivatives of a short peptide (H1) derived from residues 109 through 122 of the prion protein PrPC have been synthesized and characterized by FTIR.

Probing the effect of side chains on the conformation and stability of helical peptides via isotope-edited infrared spectroscopy.

The mechanism of helix stabilization or destabilization by different amino acids has been the subject of several experimental and theoretical studies; these studies suggest that large or bulky side chains may modulate helix stability by altering the hydration of the helix backbone. In this paper, we report a spectroscopic study to determine the effect of alanine to leucine substitutions on the conformation and solvation of specific segments of a model helical peptide. A 25-residue, alanine-rich, helical peptide [Ac-(AAAAK)(4)-AAAAY-NH(2) (AKA)] and its two leucine variants [Ac-LLLLK-(AAAAK)(3)-AAAAY-NH(2) (LKA) and Ac-(AAAAK)(4)-LLLLY-NH(2) (AKL)] were characterized by infrared (IR) and electronic circular dichroism (ECD) spectroscopies.