Quinary structure alters protein folding landscapes Public Deposited

Downloadable Content

Download PDF
Last Modified
  • March 22, 2019
  • Cohen, Rachel
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • Most knowledge of protein chemistry is derived from experiments performed in dilute, buffered solutions. Although such experiments provide essential information, proteins function in the crowded and complex cellular environment, which imposes an additional level of structure. This quinary structure comprises the transient interactions between macromolecules that provides organization and compartmentalization inside cells. I have used in-cell NMR spectroscopy to characterize quinary structure, and have shown that globular protein stability is affected by quinary interactions involving both the folded state and the unfolded ensemble. Chapter 1 reviews the history of quinary structure in the context of metabolic pathways, and the technological advances that have yielded recent insight into protein behavior in living cells. In Chapter 2, I use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells to show that quinary interactions influence the quality of in-cell 15N–1H HSQC NMR spectra. In Chapter 3, I quantify the pH-dependence of GB1 stability in cells. At neutral pH, GB1 stability in cells is comparable to that in buffer. As the pH decreases, the increased number of attractive interactions between E. coli proteins and GB1 destabilizes GB1 relative to buffer alone. I conclude that electrostatic interactions involving surface residues of the folded state contribute to quinary structure. Chapter 4 shows that quinary structure can also affect the unfolded state ensemble. It has been known for many years that the unfolded ensemble of GB1 is stabilized by a non-native hydrophobic staple. Exploiting this idea, I made several mutations that do not change the folded state of GB1, but have a large effect on its stability in buffer. These effects are severely attenuated in cells, demonstrating that the cellular environment can remodel the unfolded ensemble. My work shows that there is more to protein stability than a well-packed hydrophobic core; the key to understanding protein behavior in nature lies in quinary structure.
Date of publication
Resource type
Rights statement
  • In Copyright
  • Pielak, Gary J.
  • Lee, Andrew
  • Riordan, John
  • Brustad, Eric
  • Redinbo, Matthew R.
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017

This work has no parents.