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. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins. C ataracts are a protein misfolding disease caused by the aggregation of lens crystallin proteins into insoluble deposits that blur vision (1, 2). Because these proteins are not regenerated, damage from UV radiation, oxidative stress, and other chemical modifications accumulates with time (1, 2). As a result, over 50% of the population over 55 develops age-related cataracts (2). Additionally, numerous mutations that destabilize crystallin protein folds are linked to inherited and juvenile-onset cataracts (1). Although the causative factors associated with this disease are known, the structures of the aggregates and the mechanisms by which they form are unknown.Like other protein aggregation diseases such as type II diabetes mellitus and Alzheimer's disease, the molecular structures of proteins in cataracts are difficult to determine. Atomic-level structures have been obtained for some amyloid aggregates of peptides using NMR spectroscopy (3, 4) and X-ray crystallography (5). However, the most widely used techniques for studying aggregate structures and aggregation mechanisms are circular dichroism spectroscopy, fluorescence spectroscopy, and transmission electron microscopy, which provide little detailed structural information. Two-dimensional (2D) IR spectroscopy is emerging as an important tool for studying protein aggregates such as amyloid fibrils (6-8) because it provides bond-by-bond structural resolution on kinetically evolving samples (6, 8-10). Two-dimensional IR spectroscopy probes secondary structure through cross peak couplings and solvent exposure through 2D lineshapes. Its bond-specific structural resolution comes from isotope labeling. Mech...

Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form ␣-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the ␣-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis.

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. The sign of the coupling constant remained consistent for larger label separation (with the exception of i, i + 4) and for more labels with uniform separation. Such effects confirm that the isotopically labeled group vibrations are essentially only coupled to each other and are effectively uncoupled from those of the unlabeled groups. This development confirms the utility of isotopic labels for site-specific structural studies with vibrational spectra. Observed spectral effects cannot be explained by considering only transition dipole coupling (TDC) between amide oscillators, particularly for smaller label separations, but the TDC and ab initio predicted couplings roughly converge at large separation.

The two-dimensional infrared spectra of a series of doubly isotopically substituted 25-residue R-helices were measured with femtosecond three pulse infrared time domain interferometry. The insertion of 13 Cd 16 O and 13 Cd 18 O labels at known residues on the helix permitted the vibrational couplings between different amide I′ modes separated by one, two, and three residues to be measured. The 2D IR signal of one residue in 25 was readily studied, confirming this approach is applicable to labeled proteins. We identified the couplings between each pair of isotopomer levels and between them and the helix exciton band states: the 2D IR spectra proved that the amide vibrations of the R-helix are delocalized. Cross-peaks, originating from the coupling of the isotopomer pairs, were systematically analyzed. Besides the separated pair modeling and second-order perturbation theory estimates, the experimental results were compared in detail with a full matrix diagonalization simulation based on averaged Hamiltonian matrices that represent the amide I′ vibrator's one-and two-exciton states. The main features of the 2D IR spectra could be predicted by this modeling. The experimental results were in good agreement with a set of couplings that were derived from transition chargetransition charge interactions for all but the nearest neighbors, for which the coupling is more influenced by through-bond interactions between the adjacent amide groups. The possible ranges of the magnitudes of the three largest coupling constants β 12 , β 13 , and β 14 were explored by various approaches to be within a few cm -1 accuracy of a preferred set of absolute values and their associated error bars: |β 12 | ) 8.5 ( 1.8, |β 13 | ) 5.4 ( 1.0, and |β 14 | ) 6.6 ( 0.8 cm -1 . The signs were independently indicated to be β 12 > 0, β 13 < 0, and β 14 < 0. Recently, dual frequency phase-locked 2D IR of peptides have † Part of the special issue "Gerald Small Festschrift".

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. Thus, site-specific structural information can be obtained by sequential, systematic labeling of the backbone. This method of isotope-edited infrared spectroscopy is a tool for obtaining medium-resolution information about the backbone conformation and dynamics. This tool has been used to dissect the conformation and dynamics of alpha helices and amyloid aggregates, where the versatility of possible sampling with infrared spectroscopy is well-suited for studies of large-protein aggregates.

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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. The alignment of strands proceeds via a rapid interchange from one sheet to another. This realignment of the peptide strands into a more favorable registry may have important implications for therapeutics since previous work has shown that well aligned beta-sheets form more stable amyloid fibrils.

Many neurodegenerative diseases are characterized by the accumulation of amyloid fibers in the brain, which can occur when a protein misfolds into an extended ␤-sheet conformation. The nucleation of these ␤-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 ␤-strands of the peptide have adopted their equilibrium antiparallel ␤-sheet configuration with residue 117 in register across all strands. In this article, we present the kinetic details of the realignment of these ␤-strands from their fastformed nonequilibrium structure, which has no regular register of the strands, into the more ordered ␤-sheets capable of aggregating into stable fibers. This process is likely the nucleating step toward the formation of stable fibers. Isotope-edited IR spectroscopy is used to monitor the alignment of the ␤-strands by the introduction of a 13 C-labeled carbonyl at residue 117. Nonexponential kinetics is observed, with a complex dependence on concentration. The results are consistent with a mechanism in which the ␤-sheet realigns by both the repeated detachment and annealing of strands in solution and reptation of polypeptide strands within an aggregate.IR spectroscopy ͉ peptide aggregation ͉ isotope-edited ͉ prion peptide ͉ amyloid fiber T he misfolding of proteins into a ␤-sheet configuration and the subsequent aggregation of these ␤-sheets is associated with many neurological disorders, including Alzheimer's, Huntington's, and Creutzfeldt-Jakob disease (1). These diseases are associated with the presence of large amyloid plaques, which contain fibrous aggregates of protein. Several detailed models for the mechanism of amyloid growth have been proposed, but all involve a conformational change in the protein that leads to the formation of soluble oligomers, which eventually nucleate the growth of larger fibers. With several diseases, including Alzheimer's and Parkinson's, the oligomeric intermediates may be the primary cytotoxic species (2-4).The soluble oligomeric intermediates are difficult to isolate and, because of their large size and dynamic nature, difficult to characterize with traditional biophysical techniques. For these reasons, many studies aimed at studying the aggregation process use small peptides, derived from full-length proteins of interest, which also show amyloidogenic behavior. Among the peptides studied extensively include the NFGAIL sequence from the islet amyloid polypeptide (5-7), fragments of the prion protein that include the AGAAAAGA amyloidogenic region (8-12), and many different fragments of the Alzheimer's A␤ peptide (13)(14)(15)(16)(17)(18). In simulations of these peptides, ensembles of ␤-sheet oligomers are initially formed, including species that have a non-native hydrogen-bonding registry or mix parallel and ant...