Fluorescence resonance energy transfer (FRET) is a method used to elucidate changes in a protein’s structure (conformational changes). FRET works by measuring distances between amino acids in the presence and absence of ligand. The distance is measured by the highly distance dependent degree of energy transfer between an acceptor and a donor, typically two fluorophores which overlap in absorption and emission spectra. FRET is limited by the range of distances at which it is accurate, incomplete incorporation of acceptors, and its ease of use. The technique we will use to improve FRET is click chemistry. Click chemistry is a set of highly specific reactions that allow for the selective addition of desired functional groups to a target of interest. Our goal is to determine if FRET will be more sensitive to smaller ranges with click chemistry, which would increase measurement accuracy of conformational changes. To determine if click chemistry can be used with FRET, we first isolate maltose binding protein (MBP) with a non-canonical amino acid (ncAA) introduced at our site of interest from cells. The role of the ncAA is to “click”, or undergo a linking reaction with a specific functional group, with our selected fluorophore to ensure its attachment to the site of interest. Our modified protein will be incubated with our fluorophore, and FRET will be done on the protein under different conditions in order to test if our modified FRET works. If this method works, we expect to see distance dependent quenching of the fluorophore which would indicate that 1) the method works and 2) would serve to establish a benchmark for future experiments with different proteins. Combining click chemistry with FRET could be a powerful tool for measuring conformational dynamics in proteins.

In response to various stimuli, proteins undergo changes in shape (conformational changes) that are essential to their function in cell signalling, enzyme activity and many other crucial biological processes. Previous studies have shown that intramolecular distance changes associated with the conformational dynamics of proteins can be probed using transition-metal-ion-fluorescence-resonance-energy-transfer (tmFRET), in which a donor fluorophore transfers light energy to an acceptor metal ion in a steeply distance-dependent manner. tmFRET is both highly sensitive and highly specific: each unique donor-acceptor pair can only measure a limited range of distances reliably. Here, we expand the utility of tmFRET by employing new strategies to create a library of unique donor-acceptor pairs each sensitive to a different range of distances.To showcase the accuracy and precision of our measurements, we use a maltose-binding-protein (MBP) model system, which undergoes a well-studied conformational change upon the binding of maltose. In order to label MBP with Cu(II) ions that act as tmFRET acceptors, we utilize macrocyclic chelator-maleimides, which simultaneously coordinate Cu(II) and covalently modify a cysteine introduced into MBP. Using amber codon suppression, we introduce fluorescent non-canonical amino acids (ncAA’s) into MBP to act as tmFRET donors. Our fluorescence measurements show that tmFRET between the ncAA donor and chelated-Cu(II) acceptor accurately reports MBP’s intramolecular distance changes. We also show that by varying the exact structure of the molecule that chelates Cu(II) we can tune the sensitivity of tmFRET to distances ranging from 0.8-1.8 nanometers. Thus, this chelator-maleimide approach to labelling proteins with tmFRET acceptors could prove to be a powerful tool for researchers studying conformational motions of that scale.

Poor sleep is a common complaint during pregnancy that is associated with worse maternal and fetal outcomes including increased depression symptoms before and after pregnancy. Mindfulness-based interventions are effective in improving sleep, but little is known about the relationship between mindfulness and sleep during pregnancy. This cross-sectional study explores the associations among mindfulness, objective and subjective sleep parameters, and mood (depression, anxiety, and positive affect) during pregnancy. Participants are between 12 weeks and 28 weeks gestation, enrolled in my mentor’s pilot randomized controlled trial, Online Prenatal Trial in Mindfulness Sleep Management (OPTIMISM). Data include self-report questionnaires, actigraphy, and daily sleep diaries collected at baseline before receiving a sleep intervention. We hypothesize that mindfulness and sleep quality are positively correlated, whereas mindfulness and mood symptoms are negatively correlated. This study's findings would support mindfulness as a potential non-pharmacologic intervention to improve sleep during pregnancy.