Die Kettendynamik eines entfalteten Proteins
Zusammenfassung der Projektergebnisse
The denatured state of proteins is an elusive ensemble of conformers that can spontaneously fold into a functional three‐dimensional structure. If folding goes wrong, misfolded species can lead to formation of toxic aggregates and disease. Denatured states of ultrafast folding proteins are of particular interest in mechanistic studies because they are energetically close to the transition state, the kinetic bottleneck of the folding reaction. The denatured state populated under physiological conditions (D phys) is a transient species that rapidly converts in the folded structure and thus highly elusive. Here, we generated D phys of the helical protein domain BBL, which folds within microseconds, by a single point mutation and investigated structure and dynamics using a combined experimental/computational approach. Site‐directed mutagenesis in combination with far‐UV circular dichroism spectroscopy and single‐molecule fluorescence fluctuation analysis was applied to characterize secondary structure and main chain dynamics. We found significant amount of beta‐sheet and little helix in an otherwise largely unfolded ensemble, which was surprising given that the native fold of BBL is mainly helical. Residual structure was insensitive to 20 point mutations introduced individually along the entire sequence area deleting side chains that importantly stabilize the fold. Circular dichroism spectra superimposed and nanosecond loop closure kinetics remained virtually unchanged. Experimental work was complemented by all‐atom replica‐exchange molecular dynamics (REMD) and constant‐temperature MD simulations that provided molecular details. Simulations showed a highly collapsed, hydrogen‐bonded globule that contained beta‐sheets evenly distributed along the sequence and few nucleating helices, in agreement with experimental results. How does a protein that forms non‐native beta‐structure in its denatured state so rapidly fold into a helical domain? Recent theoretical work suggests the presence of a pathway connecting beta‐sheet and alpha‐helix conformations in the Ramachandran plot under folding conditions once hydrogen‐bonding constraints are included. We found that, indeed, the free energy surface of D phys in Ramachandran space contained a low‐energy pathway between the beta‐sheet and the alpha‐helix basin facilitating ultrafast conversion of conformers. Non‐native beta‐structure in protein denatured states is not only precursor for formation of toxic aggregates but can also nucleate native helix very early in the folding of globular domains.
Projektbezogene Publikationen (Auswahl)
- β-Structure within the denatured state of the helical protein domain BBL, J. Mol. Biol. 2015, 427, 3166- 3176
Lipi Thukral, Simone Schwarze, Isabella Daidone, and Hannes Neuweiler
(Siehe online unter https://doi.org/10.1016/j.jmb.2015.08.007)
