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...

While amyloid formation has been implicated in the pathology of over twenty human diseases, the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labeling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin, the peptide responsible for islet amyloid formation in type 2 diabetes, with a known inhibitor, rat amylin. Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 hours after mixing rat amylin blocks the N-terminal β-sheet instead. At 24 hours after mixing, rat amylin blocks neither β-sheet and forms its own β-sheet most likely on the outside of the human fibrils. This is striking because rat amylin is natively disordered and not previously known to form amyloid β-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for 13 C= 18 O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide form type 2 diabetes.

We report a structural study on the membrane binding of ovispirin using 2D IR line shape analysis, isotope labeling, and molecular dynamics simulations. Ovispirin is an antibiotic polypeptide that binds to the surfaces of membranes as an alpha-helix. By resolving individual backbone vibrational modes (amide I) using 1-(13)C=(18)O labeling, we measured the 2D IR line shapes for 15 of the 18 residues in this peptide. A comparison of the line shapes reveals an oscillation in the inhomogeneous line width that has a period equal to that of an alpha-helix (3.6 amino acids). The periodic trend is caused by the asymmetric environment of the membrane bilayer that exposes one face of the alpha-helix to much stronger environmental electrostatic forces than the other. We compare our experimental results to 2D IR line shapes calculated using the lowest free energy structure identified from molecular dynamics simulations. These simulations predict a periodic trend similar to the experiment and lead us to conclude that ovispirin lies in the membrane just below the headgroups, is tilted, and may be kinked. Besides providing insight into the antibiotic mechanism of ovispirin, our procedure provides an infrared method for studying peptide and protein structures that relies on the natural vibrational modes of the backbone. It is a complementary method to other techniques that utilize line shapes, such as fluorescence, NMR, and ESR spectroscopies, because it does not require mutations, the spectra can be quantitatively simulated using molecular dynamics, and the technique can be applied to difficult-to-study systems like ion channels, aggregated proteins, and kinetically evolving systems.

The aggregation of human amylin to form amyloid contributes to islet β-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulations and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a “W” pattern, as a function of residue number. We show that large line widths result from substantial structural disorder, and that this pattern is indicative of the stable secondary structure of the two β-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.