An undergraduate laboratory exercise is described to demonstrate the biochemical applications of electron paramagnetic resonance (EPR) spectroscopy. The β93 cysteine residue of hemoglobin is labeled by the covalent binding of 3-maleimido-proxyl (5-MSL) and 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate (MTSL), respectively. The excess spin label is removed by gel-exclusion chromatography. Changes in the mobility of the reporter groups attached to the protein are monitored by EPR spectroscopy. While the spectral parameters of the rigidly attached 5-MSL provide information on the rotation of the whole spin labeled protein, MTSL bound by a more flexible linkage describes the local environment of the cysteine residue in the interior of the protein structure. Students can study the known crystal structure of hemoglobin in comparison to the results they obtain by analyzing the EPR spectra. Overall, the exercise introduces them to laboratory techniques such as protein labeling, gel filtration, EPR spectroscopy, as well as familiarizes them with the online Protein Data Bank as a research resource and PyMOL software as a structure visualization tool. © 2013 by The International Union of Biochemistry and Molecular Biology, 41(3):156–162, 2013

A broad group of structurally diverse small organofluorine compounds were synthesized and evaluated as inhibitors of β-amyloid (Aβ) self-assembly. The main goal was to generate a diverse library of compounds with the same functional group and to observe general structural features that characterize inhibitors of Aβ oligomer and fibril formation, ultimately identifying structures for further focused inhibitor design. The common structural motifs in these compounds are CF₃-C-OH and CF₃-C-NH groups that were proposed to be binding units in our previous studies. A broad range of potential small-molecule inhibitors were synthesized by combining various carbocyclic and heteroaromatic rings with an array of substituents, generating a total of 106 molecules. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy for monitoring fibril formation, biotinyl Aβ_1–42 single-site streptavidin-based assays for observing oligomer formation, and atomic force microscopy for morphological studies. These assays revealed a number of structures that show significant inhibition against either Aβ fibril or oligomer formation. A detailed analysis of the structure–activity relationship of anti-fibril and -oligomer properties is provided. These data present further experimental evidence for the distinct nature of fibril versus oligomer formation and indicate that the interaction of the Aβ peptide with chiral small molecules is not stereospecific in nature.

Molecular docking is a frequently used method in structure-based rational drug design. It is used for evaluating the complex formation of small ligands with large biomolecules, predicting the strength of the bonding forces and finding the best geometrical arrangements. The major goal of this advanced undergraduate biochemistry laboratory exercise is to illustrate the importance and application of this tool. Students carry out the computational modeling of the interaction of acetylcholinesterase and its inhibitor, tacrine, and learn about the concepts of protein structure, enzyme-inhibitor interactions, intermolecular forces, and role of molecular design in drug-development.