"Invisible" conformers of an antifungal disulfide protein revealed by constrained cold and heat unfolding, CEST-NMR experiments, and molecular dynamics calculations.

Ádám Fizil,Terézia Barna,Gyula Batta,Florentine Marx,Zoltán Gáspári

doi:10.1002/chem.201404879

Abstract

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide-rich protein. In addition, sensitive 15N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction.

Highlights

The mode of action of globular proteins is conventionally explained by their “functional” native structure
Internal dynamics at various timescales has emerged as a key additional determinant of molecular function, ranging from fluctuations about a well-defined state to substantial conformational freedom exemplified by intrinsically disordered proteins (IDPs).[1,2,3,4,5,6,7,8]
We performed a detailed NMR study on the hot and cold unfolding of PAF and performed experiments to identify hidden conformations that are in slow to intermediate exchange with the major observable form. This highly stable and disulfide-constrained protein remains largely structured under all conditions we applied, a number of different conformers are still likely to be present in dynamic equilibrium with each other

Summary

Full Paper

& Conformation Analysis “Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding, CEST-NMR Experiments, and Molecular Dynamics Calculations. dμm Fizil,[a] Zoltμn Gμspμri,[b] TerØzia Barna,[c] Florentine Marx,[d] and Gyula Batta*[a]

Introduction

PAF does not undergo complete unfolding

Results and Discussion

Implications for NMR data interpretation and submission practice

Hidden conformers and PAF function

Experimental Section