Structure-function relationships of proteins are of significant importance in the study of biochemistry. In order for students to understand these relationships, it is essential for them to understand protein structure and, more specifically, the different conformations of proteins. With this in mind, we are developing an undergraduate biochemistry laboratory experiment to enhance student comprehension of protein conformations, redox reactions, and high-performance liquid chromatography (HPLC). Through the use of reversed-phase HPLC with a C-18 column, different conformations of lysozyme were analyzed. This 129-residue protein is comprised of a peptide chain interconnected with four disulfide bonds. Being completely folded, the native conformations of lysozyme expose their polar surfaces and thus have the shortest retention times in the column. As the denatured proteins are partially unfolded, they expose parts of their interior hydrophobic residues, allowing them to have longer retention times. Due to disulfide bond breakages and the consequent complete unfolding, the reduced proteins have the longest retention times in the column. With the promising results from the analysis of lysozyme’s conformations, we have optimized and replicated the instrumentation protocols on an additional protein. This has enabled us to not only verify the robustness of our data, but also adjust the experiment to become more suitable for students in an upper-level biochemistry laboratory course.

Protein structures analysis using reversed-phase high-performance liquid chromatography

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.