Organization of Biochemistry I and II labs

The first semester of this course is designed to introduce you to methodologies and techniques used in modern biochemistry. You will learn these methods while studying the protein of interest (POI), nucleoside kinase from Thermoplasma acidophilium (TaNK). Unlike previous lab courses, pre-canned protocols will not be provided. This laboratory is designed for you to learn to explore, research, and investigate science independently. You are expected to read textbooks, literature, and instructions before lectures and lab sessions. The lab and lecture are designed to help you teach yourself; they are not intended to passively feed you information.

The assigned protein was studied previously by a UVA Chemistry alum. She began her work by expressing the protein from a plasmid provided by the JCSG (Ta0880; PDB id 3bf5). The protein’s structure was known and the function was annotated based on that structure. Enzymatic assays showed that the protein did not have the annotated function (ribokinase) but studies of the fold highlighted the structural similarity to nucleoside kinases (NKs). Subsequent enzymatic assays determined the protein to be a broad specificity nucleoside kinase. 

You will study the protein’s structure and active site in order to understand what amino acid (AA) residues in the active site confer specificity for particular substrates. You will then propose and generate a site-specific AA mutation that you predict would alter that specificity. In the spring semester (CHEM4421), you will examine the function of TaNK and the mutant that you create in the fall semester. All of the material you study in the fall semester is relevant to the spring semester. This guide and the data you acquire are the information that you will heavily rely on in the second semester. In the spring semester, you will design and execute your own experiments in order to characterize how the mutation affects TaNK. 

All of the above is genuine (original) research, and in this way may differ from the experiments that you performed in your previous undergraduate lab courses. Any characterization that you accomplish for your TaNK mutant should be considered genuine scientific progress (such as in the publications you read). If your data and results are reliable and thorough, they may be published or disseminated via databases (e.g., TOPSAN) with you as an author.

Performing research of this sort is an excellent way to learn how to approach a question scientifically. However, because it is truly original research, results are not guaranteed. Your grade will not rely on you getting a specific set of data. However, regardless of how well your experiments work, you will be responsible for making reasonable and logical choices in your approach, and for making a concerted and sustained effort to move your project along.