The use of single tryptophan variants to study protein folding and stability
MetadataShow full item record
Studies on the kinetics of protein folding of the histidine-containing phosphocarrier protein (HPr) from the thermophile Bacillus stearothermophilus (Bst) will contribute much to the understanding of the origins of its enhanced thermal stability. Although circular dichroism (CD) is available to us as a method by which to study the unfolding properties of HPr, the more effective method of fluorescence spectroscopy improves on the high signal-to-noise ratio of CD. Tryptophan is the best amino acid to use as a probe by which to monitor unfolding by fluorescence; however, wild type BstHPr contains no tryptophan residues. In order to utilize this technique, three tryptophan-containing variants of HPr (F6W, F29W, and Y37W) were designed and expressed in hopes of finding a variant protein which possesses similar properties to wild type HPr so that it may be used as a model. The usefulness of these probes were analyzed by fluorescence emission scans, and it was determined that each of the three residues acted as good probes by which to monitor the folding and unfolding of HPr by fluorescence. Subsequently, experiments were performed where urea denaturation was monitored by circular dichroism and fluorescence. [Delta]G and m-values for each variant protein were obtained by analysis of the resultant urea denaturation curves. Results indicated that Y37W has the closest [delta]G of stabilization and m-value to the wild type protein, suggesting that it can be used as a model protein for kinetic studies of the folding mechanisms of HPr.
DescriptionDue to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to firstname.lastname@example.org, referencing the URI of the item.
Includes bibliographical references (leaf 22).
Dulin, Jennifer Natalie (2004). The use of single tryptophan variants to study protein folding and stability. Texas A&M University. Available electronically from