Etablierung eines Systems aus Cysteinmutanten der Phosphoglycerat-Kinase für Entfaltungsstudien mit Einzelmolekül-FRET

Proteins maintain life through a multitude of tasks within biological systems. To gain their functionality, it is crucial that proteins obtain their native structure which is hidden within their amino acid sequence. Deciphering how the amino acid code translates into 3D structures is the key to understand how proteins really work. In this context, the twodomain protein phosphoglycerate kinase (PGK) has proven to be an excellent model for multi-domain proteins. It is known that both domains of PGK interact during folding. The N-terminal domain only gains its native structure in presence of the C-terminal domain. The C-domain also folds individually but the process is facilitated by the Ndomain. In addition, at least one intermediate state is involved. A detailed picture of the folding pathway of PGK and to what extent intermediates are populated is still missing. It is challenging to unravel the mechanisms of tertiary structure formation, especially since subpopulations are hard to identify with ensemble methods. To avoid averaging over all conformations, single-molecule methods are a promising tool to distinguish and quantify intermediate states. Here, a set of three yPGK cysteine variants for site-specific labeling with fluorescent dyes for single molecule fluorescence resonance energy transfer (FRET) was established. This system is designed to follow motions in between and within the individual domains displayed by distance changes of fluorophores during unfolding transitions under denaturing conditions. It was verified that secondary and tertiary structures were not considerably affected by cysteine mutations applying circular dichroism (CD) spectroscopy and dynamic light scattering (DLS). In addition, all PGK cysteine mutants were catalytically active. The native states of the double labeled PGK variants were thoroughly characterized by fluorescence correlation spectroscopy (FCS) and single molecule FRET. Thus, a quality test to proof the suitability of yPGK variants for single-molecule FRET studies was established. First unfolding experiments (applying GndHCl for chemical denaturation) under equilibrium conditions were performed for all variants of the system. Unfolding of the inter-domain mutant, carrying a fluorophor in each of its domains, follows the classical two-state model. In contrast, the intra-domain mutants show more complex unfolding patterns. The results indicate that the N-domain forms a compact intermediate. The C-domain seems to stay locally compact even beyond the critical range of denaturant concentrations. Further studies based on the optimized sample preparation and the established quality test will provide detailed insights into the unfolding pattern of yPGK and its domains. Promising candidates to extend the proposed system were already identified

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates

The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation

Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions

The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye–dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil–globule transition