Unfolding Kinetics and Thermodynamic Stability of Type Three Secretion System Effectors

Weible, Ezra M.

Undergraduate Honors Thesis

Unfolding Kinetics and Thermodynamic Stability of Type Three Secretion System Effectors Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/6q182m84z

Abstract

The Type Three Secretion System (T3SS) of Gram-negative bacteria is a syringe-like protein complex that injects virulence effector proteins directly into the host cell, facilitating bacterial infection. However, the needle is too small to inject fully folded proteins. We propose that the T3SS mechanically unfolds effectors before injection and that effectors evolved to be thermodynamically stabile but mechanically labile, aligning with previous data for effectors SptP and SopE2. However, it is also possible that effectors have evolved to have fast unfolding kinetics (intrinsic rates of unfolding). To test this possibility, the intrinsic rates of unfolding were measured for SptP and SopE2 resulting in ln(k_unfold) values of -5.8 ± 0.3 and -3.7 ± 0.8. These rates are typical for proteins with mixed secondary folds, indicating that effectors have not evolved unusually fast intrinsic unfolding kinetics.

Furthermore, we note that effectors display extreme sequence divergence from Non-Effector Homolog (NEH) proteins, which we propose is due to the evolutionary pressure to be thermodynamically stable but mechanically labile. To test this hypothesis, we aim to compare the thermodynamic and mechanical stability of effector and NEH proteins. Here I report that the apparent thermodynamic folding stability (ΔG^o_unfolding) of the NEH Protealysin, a homolog of the T3SS effector NleC, is 4.1±0.5 kcal/mol. This is comparable to other globular proteins found in the literature, including SptP and SopE2. This provides evidence for our hypothesis and against the widely accepted model that effectors are thermodynamically unstable.

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