Trp RNA-Binding Attenuation Protein: Modifying Symmetry and Stability of a Circular Oligomer

Abstract

BackgroundSubunit number is amongst the most important structural parameters that determine size, symmetry and geometry of a circular protein oligomer. The L-tryptophan biosynthesis regulator, TRAP, present in several Bacilli, is a good model system for investigating determinants of the oligomeric state. A short segment of C-terminal residues defines whether TRAP forms an 11-mer or 12-mer assembly. To understand which oligomeric state is more stable, we examine the stability of several wild type and mutant TRAP proteins.Methodology/Principal FindingsAmong the wild type B. stearothermophilus, B. halodurans and B. subtilis TRAP, we find that the former is the most stable whilst the latter is the least. Thermal stability of all TRAP is shown to increase with L-tryptophan concentration. We also find that mutant TRAP molecules that are truncated at the C-terminus - and hence induced to form 12-mers, distinct from their 11-mer wild type counterparts - have increased melting temperatures. We show that the same effect can be achieved by a point mutation S72N at a subunit interface, which leads to exclusion of C-terminal residues from the interface. Our findings are supported by dye-based scanning fluorimetry, CD spectroscopy, and by crystal structure and mass spectrometry analysis of the B. subtilis S72N TRAP.Conclusions/SignificanceWe conclude that the oligomeric state of a circular protein can be changed by introducing a point mutation at a subunit interface. Exclusion (or deletion) of the C-terminus from the subunit interface has a major impact on properties of TRAP oligomers, making them more stable, and we argue that the cause of these changes is the altered oligomeric state. The more stable TRAP oligomers could be used in potential applications of TRAP in bionanotechnology.

Tuning the Size and Properties of ClyA Nanopores Assisted by Directed Evolution

“…Although alternative folded ClyA structures cannot be excluded, the observation that ClyA oligomers with a slower electrophoretic migration form nanopores with higher conductance in lipid bilayers suggests that the three ClyA types might correspond to oligomers with different subunit stoichiometry. This is not necessarily surprising given that high order symmetrical oligomeric structures are often permissive with respect to subunit stoichiometry, 37,38 and the exact stoichiometry of E. coli ClyA (~90% sequence identity to Salmonella typhi ClyA) oligomerisation is controversial: the crystal structure (PDB_ID: 2WCD) revealed a dodecamer 36 , while earlier cryo-EM studies revealed nanopores with 8 39 or 13 40 subunits. We hypothesize that the major band on the BN-PAGE corresponding to Type I ClyA most likely represents the 12mer of the crystal structure, while the band corresponding to Type II ClyA might be the 13mer observed in earlier cryo-EM studies.…”

Population Distributions from Native Mass Spectrometry Titrations Reveal Nearest-Neighbor Cooperativity in the Ring-Shaped Oligomeric Protein TRAP

“…(MW 101,363 Da with normalized intensity as high as 10.5% compared to the 11-mer); although uncommon, small populations of TRAP 12-mers have been observed in mass spectrometry experiments 38 as well as a few solution experiments, 49,50 and in crystals. 33 In addition, we observed signals corresponding to trace dimers of TRAP11 ("double-donuts"), in agreement with previous solution experiments.…”

The near‐symmetry of proteins

“…Proteins symmetry research continuous to be a central theme in structural biochemistry, and some recent examples are collected in Refs. . Understanding the abundance of this structural feature—symmetry—has received much attention, because at first glance it is not clear why this feature is important for proteins function, that is, why has symmetric clustering evolved at all.…”