Probing protein structure and aggregation in complex environments with 2D IR spectroscopy

The goal of our proposed research program is to develop new spectroscopic methods capable of providing enhanced insight into protein structure and dynamics within complex environments that impact human health. We will apply these methods to study structural changes induced by protein-protein and protein-surface interactions under conditions that are not accessible by other experimental approaches. Our first research direction focuses on advancing the use of unnatural amino acids (UAAs) beyond probes of local environment by taking advantage of the unique spectral features provided by two-dimensional infrared (2D IR) spectroscopy to measure vibrational couplings between UAA labels. These couplings depend sensitively on the distance and relative orientation between UAA vibrational modes and thus can be used to map protein structure. We will select UAAs modified with functional groups that have vibrational modes within a biologically transparent region of the infrared spectrum, allowing us to probe the structure of protein complexes and aggregates in biological media. We will use the Alzheimer’s β-amyloid (Aβ) protein, the most studied self-assembling protein, in cerebrospinal fluid as a prototypical system to develop and refine our approach into a broadly applicable method that will enable the study of dynamic protein interactions that result in formation of protein complexes and aggregates involved in a range of diseases. The Aβ studies will also allow us to bridge the gap between in vitro aggregation studies and ex vivo fibril structures and gain unprecedented new insight into physiologically relevant self-assembly pathways in Alzheimer’s disease. Our second research direction aims to understand, for the first time, the detailed residue-level changes to protein structure that occur when proteins interact with nanoparticle (NP) surfaces. NPs are ubiquitous in our lives and are increasingly being considered for biomedical applications. However, the effect of nanomaterials on living systems remains unclear. One concern is that proteins readily adsorb onto the surfaces of NPs, which can result in changes to protein structure and thus function. Our initial studies will examine the differing effects of NPs on the secondary structure of model peptides and lysozyme, first with metallic NPs as a model system to understand the effects of NP size and concentration, and then with silica NPs to examine the role of surface chemistry in affecting structural changes. Machine learning-based approaches to enhance the sensitivity of 2D IR spectroscopy to site-specific labels will be developed to further improve structural resolution. Ultimately, these studies will be expanded to understand the interactions between a broader range of both nanomaterials and proteins in biologically relevant media.