Novel Isotope Labeling Strategies for NMR-Based Structure Determination of High Molecular Weight Proteins

• Author / Creator

  Danmaliki, Gaddafi

• Solution NMR studies of high molecular weight proteins suffer from inadequate side-chain information and rapid signal decay. Selective 1H, 13CH3-methyl labelling of perdeuterated proteins has been the standard approach addressing these limitations but suffers from a lack of information about the non-methyl-containing amino acids. Here, we present methods for introducing additional isolated 1H-12C groups into proteins using inexpensive metabolic precursors and inhibitors.
  Using fumarate as a carbon source for Escherichia coli (E. coli) in D2O allows for stereospecific incorporation of 1H at beta2 and 2H at beta3 positions of the oxaloacetate family of amino acids (Asp, Asn, Met, and Lys). We demonstrate an efficient and inexpensive synthetic protocol for producing phenylpyruvate, hydroxyphenylpyruvate, and anthranilate precursors for labelling Phe (d, z), Tyr (d), and Trp (d, h, e), respectively. We labelled the imidazole ring of histidine by supplementing the E. coli growth medium with the unlabelled amino acid.
  The 1H-12C groups introduced in eight non-methyl-containing amino acids complement methyl groups and can be connected to 1H-15N amide positions using multi-dimensional through-space NOESY experiments. We demonstrate the utility of our approach by labelling the outer membrane protein PagP in DPC detergent micelles (>50 kDa), yielding rich new structural restraints for chemical shift assignments and high-resolution structure determination.
  Solution NMR is the ideal technique for probing protein motion over various timescales. Most NMR studies frequently focus on quantifying only the backbone dynamics by measuring 15N relaxation properties in uniformly labelled proteins. Side chain motion also plays a pivotal role in many biological processes. However, characterizing side chain dynamics remains difficult for solution NMR. We introduce a new method for assessing protein side-chain motion using 1H NMR relaxation rates, extending the dynamic analysis to exciting unstudied protein sites.

• Subjects / Keywords

  • Solution NMR
  • Isotope Labeling
  • Selective Methyl Group Protonation
  • High Molecular Weight Proteins
  • Side Chain Dynamics

• Graduation date

  Spring 2023

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-txn7-hy47

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.