Experimental Design

CHEM 4411 and 4421: Biological Chemistry Lab

Measuring Activity using Assays

Sterics of Conserved Residue K422 inThermotoga maritima Phosphoglucose Isomerase Affect Catalytic Activity

Dr. Carol Price, Andrew Spira

Course Summary

This was a year-long lab that focused on the design and execution of a group research project. The aim of this research was to investigate the structure-function dynamics of the active site on enzyme activity. I tested the effect of a point mutation (K422H) on the activity of phosphoglucose isomerase, a protein in the thermophilic bacteria, Thermotoga maritima, or TmaPGI.

Skills

Scientific research: methodology, organization, collaboration

Analytical techniques

• Protein Quantitation (NanoDrop A₂₈₀)
• Assay (Epoch 2 Microplate reader)
• Software: OriginPro 7.5

Role of steric hindrance at the active site

I concluded sterics may have a great impact on active site function for TmaPGI. The point site mutation focused on increasing the catalytic efficiency of site 422, a basic residue located within the active site, in hopes of increasing the overall catalytic activity of the protein.

During the analysis stage of this project, my group compared results from the K422H mutation to similar groups focused on site 422 mutations. We identified a relationship between steric bulk at the residue 422 site and subsequent effect on catalyic efficiency of TmaPGI: as the residue size at site 422 increases, the catalytic efficiency, or protein activity, decreases.

Experiment log for documentation

When working with a group containing five other members, prioritizing records was necessary. I ensured our work was well-documented by creating a group experiment log where all members could record the time and contents of their completed tasks.

G6PDH calculation error: testament to tenacity

The not so glamorous side of scientific research was highlighted in this course through the used methodology. Unlike previous student lab courses, this class emphasized an independent research project in all aspects, including the creation of our protocol. When calculating the needed concentrations and volumes that would be needed over the course of the project, an error was made regarding the needed amount of the substance G6PDH. This result was unfortunately carried through multiple experiments, until upon group evaluation with our professor and teaching assistant, it was discovered.

This course demonstrated my commitment to the process of collecting data. I gained valuable experience in the scientific process by consulting literature review and multiple resources at hand, including my mentors, to identify any issues that may arise when answering a given research question.

Poster

I summarized the key components of this research in a poster that was individually presented and discussed with my professor and graduate TA. My group’s poster was awarded the best overall poster by my peers and teaching figures.

Manuscript

The findings of this course were summarized in a manuscript, in total 21 pages.