There is considerable interest in uncovering the pathway of amyloid formation because the toxic properties of amyloid likely stems from prefibril intermediates and not the fully formed fibrils. Using a recently invented method of collecting 2-dimensional infrared spectra and site-specific isotope labeling, we have measured the development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyloid polypeptide (hIAPP). By monitoring the kinetics at 6 different labeled sites, we find that the peptides initially develop well-ordered structure in the region of the chain that is close to the ordered loop of the fibrils, followed by formation of the 2 parallel ␤-sheets with the N-terminal ␤-sheet likely forming before the C-terminal sheet. This experimental approach provides a detailed view of the aggregation pathway of hIAPP fibril formation as well as a general methodology for studying other amyloid forming proteins without the use of structure-perturbing labels.aggregation ͉ amylin ͉ fibers ͉ human islet amyloid polypeptide ͉ nucleation M ore than 20 different diseases are associated with proteins that form insoluble amyloid fibers (1). In large quantities, organ function is disrupted by the formation of amyloid deposits, but for several amyloid diseases, there is evidence that the toxic entities are actually prefibril intermediates (2, 3). Although they have been the focus of numerous studies, details about these critical intermediates have been elusive, mostly because it is extraordinarily difficult to obtain structural and kinetic information for amyloid aggregation. The difficulty arises because high-resolution techniques do not have the time resolution required to track the structural changes, nor can they be easily applied to aggregating systems. Optical techniques that do have sufficient time resolution, such as circular dichroism spectroscopy, provide only a low-resolution view of structure. Other techniques, like electron spin resonance and fluorescence spectroscopy, require bulky labels that can perturb the structure and dynamics. Mechanistic information is vital to understand the mechanism of protein misfolding as well as to design inhibitors that subvert the pathway of amyloid formation. What is needed is a technique with sufficient time resolution to observe intermediates, provide residue-level structural information, is nonperturbing, and, ideally, can be used to test molecular dynamics simulations.A technique that satisfies these criteria is 2D infrared (2D IR) spectroscopy when used with site-specific isotope labeling (4). We have recently demonstrated a technological approach for collecting 2D IR spectra that is particularly well-suited for studying amyloid formation (5). Our method uses a mid-IR pulse shaper to automate data collection, much like an NMR spectrometer, so that spectra can be collected quickly enough to monitor fibril kinetics on the fly. In this article, we combine this automated version...

The capability of 2D IR spectroscopy to elucidate time-evolving structures is enhanced by a programmable mid-IR pulse shaper that greatly improves the ease, speed, and accuracy of data collection. Traditional ways of collecting 2D IR spectra are difficult to implement, cause distorted peak shapes, and result in poor time resolution and/or phase problems. We report on several methods for collecting 2D IR spectra by using a computer-controlled germanium acoustooptic modulator that overcomes the above problems. The accuracy and resolution of each method is evaluated by using model metal carbonyl compounds that have well defined lineshapes. Furthermore, phase cycling can now be employed to largely alleviate background scatter from heterogeneous samples. With these methods in hand, we apply 2D IR spectroscopy to study the structural diversity in amyloid fibers of aggregated human islet amyloid polypeptide (hIAPP), which is involved with type 2 diabetes. The 2D IR spectra reveal that the ␤-sheet fibers have a large structural distribution, as evidenced by an inhomogeneously broadened ␤-sheet peak and strong coupling to random coil conformations. Structural diversity is an important characteristic of hIAPP because it may be that partly folded peptides cause the disease. This experiment on hIAPP is an example of how computer generation of 2D IR pulse sequences is a key step toward automating 2D IR spectroscopy, so that new pulse sequences can be implemented quickly and a diverse range of systems can be studied more easily.femtosecond spectroscopy ͉ infrared spectroscopy ͉ pulse shaping ͉ protein structure T wo-dimensional infrared (2D IR) spectroscopy is becoming a very useful tool for probing the fast structural dynamics of chemical and biological systems (1-3). Analogous in many ways to 2D NMR spectroscopy, 2D IR spectroscopy probes molecular structures by means of vibrational frequencies, couplings, and transition dipole angles (4-8). Environmental dynamics are probed through lineshape analysis (9-11). The combination of structural sensitivity and fast time resolution (fs/ps) makes this technique and its variants (3,(12)(13)(14) especially adept at monitoring the dynamics of evolving structures or the kinetics of chemical reactions (15)(16)(17)(18). Although 2D IR spectroscopy is a powerful technique, implementing it is technically challenging. In a typical 2D IR spectrometer, each pulse has its own optical path composed of several mirrors to route the beam to the sample and a mechanical delay stage that controls the time delay by changing the physical length of the optical path. All of the pulses have the same frequencies and shapes unless additional optics are added, such as an etalon to narrow a pulse bandwidth (4, 19) or a second mixing crystal to generate pulse sequences with two center frequencies (20,21). Thus, implementing even the simplest pulse sequence is a tremendous amount of work. This is in sharp contrast to 2D NMR spectroscopy, in which NMR pulse sequences are easily programmed with precisely set freq...

Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

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.

The 37-residue human islet amyloid polypeptide (hIAPP or amylin) self-assembles into fibers, the assembly of which has been associated with the disease mechanism of type II diabetes. Infrared spectroscopy in conjunction with isotope labeling is proving to be a powerful tool for studying the aggregation process of hIAPP and other amyloid forming proteins with residue specific structure and kinetic information, but the relationship between the spectroscopic observables and the structure is not fully established. We report a detailed analysis of the linear and 2D IR spectra of hIAPP fibers isotope labeled at seven different residue positions. The features of the 2D IR spectra, including the frequencies, linewidths, intensities, and polarization dependence of the diagonal and cross-peaks, rely heavily on the position of the isotope labeled residue. In order to understand how these measured parameters depend on fiber secondary and tertiary structure, we have simulated 1D and 2D IR spectra utilizing idealized structural models in addition to a recently published solid-state NMR based model of the amyloid fibril. The analysis provides a more rigorous foundation for interpreting the infrared spectra of amyloids. In addition, we demonstrate that 2D IR spectra can be employed to distinguish between residues in β-sheets versus those in turn regions, and that transitional residues between secondary structures can be identified by the suppression of their cross-peaks in 2D IR spectra. This latter approach is not limited to amyloid fibrils and will be generally useful in identifying regions of secondary structure in proteins using 2D IR spectroscopy and isotope labeling.