Probing Catalytically Relevant Dynamics in Proteins Using Solution-State NMR
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Probing Catalytically Relevant Dynamics in Proteins Using Solution-State NMR

  • Author(s): Sakhrani, Varun Vijay
  • Advisor(s): Mueller, Leonard J
  • et al.
Creative Commons 'BY-SA' version 4.0 license
Abstract

Solution-state NMR was used to obtain sequential backbone bone assignments and 15N relaxation data for two moderately sized proteins with and without their substrate analog inhibitors. Investigations of short and long-timescale dynamics were carried out using model-free analysis and molecular dynamics simulations. These studies reveal the importance of dynamics in protein loops forming the active site or taking part in allosteric communication within a complex.The overuse of penicillin antibiotics, characterized by a β‐lactam ring, has resulted in bacteria evolving to express β‐lactamase enzymes capable of hydrolyzing the β‐lactam ring rendering the drug molecules pharmacologically inactive. The dynamic properties of β-lactamases are linked to their biological function and are therefore thought to play an essential role in governing their evolution. Detailed comparisons of electrostatic and dynamic properties to reflect evolutionary trends highlight the need to procure empirical data on residue-wise dynamics for modern members of this family of enzymes. To date, the dynamics of only a small number of Class-A β-lactamases have been characterized. Toho‐1 β‐lactamase (TBL) is one such modern clinically significant Class-A member for which empirical residue-wise dynamics would benefit the design of next-generation β‐lactamase inhibitors or β‐lactamase resistant antibiotics. Our NMR findings lend empirical support to the hypothesis of dynamical duality for TBL that enables its efficient catalytic activity. The α-subunit of tryptophan synthase (αTS) has evolved in bacteria to cooperate with the beta dimer through the formation of the αββα heterodimer for the synthesis of L-tryptophan. In maize, a homolog of αTS, indole-3-glycerol phosphate lyase chloroplastic has evolved to act alone, exhibiting a turnover rate that is 1400-fold greater than free αTS. Comparisons of the X-ray crystal structures of these two proteins have led us to hypothesize that the stabilized α-L2 and α-L6 loops, along with the pre-positioning of the active site catalytic residues, are needed for fully-realized catalytic activity. Our NMR studies confirm this rigidity is absent in the free αTS and indeed, for efficient synthesis of L-tryptophan, the activity of αTS is tightly regulated both through the assembly of αTS into the αββα heterodimer and via heterotrophic allosteric interactions between the α- and β-sites.

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