Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR

Abstract

Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.

Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

“…Figure 11c states [1,31] that play important roles in protein folding [21], enzymology [72][73][74] and molecular recognition [75]. Further, the work demonstrates the utility in using a combined NMR relaxation dispersion/CSRosetta approach for studies of "invisible" excited protein states, providing structural data at a level of detail not possible using other biophysical techniques.…”

“…Although some small proteins are thought to fold by a 2-state mechanism in which the unfolded state transitions in a highly cooperative manner to the folded conformer [20] (Figure 1a), it is becoming increasingly clear that for many proteins, folding involves the formation of one or more transiently formed intermediates [1,[6][7][8][9][10]21] (Figure 1b). Sufficiently stable intermediates can be detected kinetically using stop-flow or continuous-flow techniques and proven to be onpathway by kinetic modeling [8,9,22] be populated to a level that is detectable at equilibrium by thermodynamic experiments, and in amenable cases be proven to be on-pathway by relaxation dispersion NMR, a technique which will be described in detail in later sections.…”

“…Several classes of model proteins have been studied to date, including all-beta sheet proteins such as the SH3 domain [21,52], and all-α-helical proteins such as the FF domain [1,9,53,54].…”

“…These biologically important excited states are difficult if not impossible to study by any other means. Despite the population and exchange timescale restrictions, it has already been shown that the excited states of many proteins with varied folds such as the all-α-helical apomyoglobin [103], the all--sheet Fyn SH3 domain [21] and the integral membrane protein phospholamban [104] can be studied in detail by relaxation dispersion.…”

Protein folding: Then and now

“…Besides HX methods, the site-specific structural information obtained from other NMR-based techniques such as relaxation dispersion spectroscopy [173], has also been incorporated into MD simulations to study weakly-populated folding intermediates [174].…”