Recombinant Site Specific Labeling of Proteins with IR Probes Öffentlichkeit
Cooper, Amanda Katherine (2013)
Published
Abstract
Abstract
Recombinant Site Specific Labeling of Proteins with IR Probes
By Amanda Katherine Cooper
The area of protein folding has a number of unanswered questions regarding the basic principles of how a protein achieves its native three dimensional folded conformation. Ultrafast folding proteins are small proteins with simple secondary structures that are models for kinetic studies that will provide more information on the fundamentals of how these structures form. One ultrafast folding model system with a high alpha helical content is the protein BdpA from staphylococcus. This protein has been studied both computationally and experimentally with conflicting results. The resolution of these contradictions through experiment will give insight into how alpha helical proteins form.
We have developed a labeling methodology for BdpA using a methionine auxotrophic cell line that allows for the incorporation of a heavy isotope infrared active label at a specific site of the protein. By constructing three mutant versions that incorporate this label in one of BdpA's three alpha helices, we expressed three proteins with a site in each helix for use in monitoring the kinetic parameters at the location of the label. Characterization of the proteins using infrared and circular dichroism spectroscopy shows that the point mutation introduced in different locations in each of the helices did not significantly destabilize the system, and that the label produced an isolated infrared signature that shows a temperature dependence that can be harnessed for kinetic measurements.
Preliminary kinetic measurements using temperature-jump infrared spectroscopy show that the label absorbance can be monitored for the derivation of kinetic parameters. Therefore, the insertion of these labels gives three different mutant versions of the protein with labels in various helical positions that, along with the wild type, can be compared in order to determine when the helices form in regards to the folding pathway of the protein. The derived kinetics can then be used to describe the folding pathway of the protein. By introducing minimally perturbing labels into various sites in BdpA, we can monitor site specifically using infrared spectroscopy the kinetics occurring at a highly localized site of the protein.
Table of Contents
Table of Contents
Introduction...1- 14
Protein Folding...1
BdpA Background...6
Reported Computational Results...7
Reported Experimental Results...9
Infrared Spectroscopy for Monitoring Folding...10
Specific Aims...12
Chapter 1: Protein Expression, Purification and
Characterization...15-30
Introduction...15
Materials and Methods...17
Construction of Plasmids...17
Cell Selection...18
Wild Type Expression and Purification...20
Mutant Expression and Purification...22
Removal of Hexa-Histidine Affinity Tag...23
Analysis by IR Spectroscopy...25
Results and Discussion...27
Chapter 2: Thermodynamics...31-44
Introduction...31
Materials and Methods...35
CD Spectroscopy...35
Protein and Cell Preparation...35
IR Spectroscopy...36
Infrared Data Evaluation...37
Fit Data...37
Calculation of Free Energy...38
Results and Discussion...38
Chapter 3: Kinetics...45-51
Introduction...45
Materials and Methods...47
Protein and Cell Preparation...47
Temperature Jump Spectroscopy...48
Kinetic Data Evaluation...48
Results and Discussion...49
Conclusion...52-53
References...54-55
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