Pin1, Peptidyl-prolyl cis-transisomerase NIMA interaction 1, contains two functional domains, WW domain for recognition of the peptide substrate and PPIase domain for cis-trans isomerization, linked by a flexible, intrinsically disordered region ( IDR). Recent evidence from NMR studies indicates the inter-domain migration in this bivalent protein, the mechanism of which remains enigmatic. Therefore, we characterized the inter-domain dynamics of Pin1 from multicolor single-molecule fluorescence resonance energy transfer measurements.
We measured the dynamics of Pin1 directly tethered to the surface or co-encapsulated in a lipid vesicle with a peptide substrate to observe its transient interaction with the substrate. We visualized the structural dynamics between Pin1 domains as well as their interaction with the peptide substrate in real time to reveal the temporal correlation between domain recognition and isomerization dynamics. This approach will provide a new understanding of the role of intrinsically disordered regions in coordinating the multidomain functions of proteins.
Introduction
This method is widely used in many fields to determine, to name but a few, the dynamics of RNA folding and catalysis, non-canonical DNA dynamics, ion channels or signal transduction. To solve this problem, we use a system of vesicles on FRET to see more natural dynamics of these dynamics. 2] Schematic of FRET experiment with vesicles The binding pattern on the slide (right) can distort the natural dynamics of protein domains, but the design of vesicles (left) can show naive protein dynamics.
Methods and Materials
TIRF and FRET
In a FRET measurement, nonradiative energy transfer occurs between donor and acceptor dye molecules when there is an interfacial distance that can be measured from the ratio of acceptor to total emission intensity. This is a really useful and reliable method in revealing the population distribution of distances between colors. (4). a) FRET occurs by energy transfer from the excitation of the donor as the wavelength of the acceptor emission, eventually showing the wavelength of the acceptor emission. 5] As a PPIase, Pin1 has a unique conformation. a) PPIase, unlike other amino acid residues, has a relatively higher trans form ratio due to the proline residue.
Sample preparation and purification
Since the MW of Pin1 is 20,406 Da, while the MW of maleimides cy3 and cy5 are 765.95 Da and 791.95 Da, an important step is expected to occur when the labeled protein is filtered and the free dyes begin to escape with the labeled dye. Serine amino acids at positions 98 and 126 are modified to cysteine to attach maleimide dyes, while cysteines at position 113 are modified to aspartic acids to prevent unnecessary labeling (a), (b). c) It shows the chemical reaction, how maleimide and kthiol groups form chemical bonds from cysteine residues. After the second column, from the third column, the dye concentration increased dramatically with a slight decrease in the last wash.
Based on these data, it is expected that initially two columns will contain mainly unlabeled protein with labeled protein, while other columns will show that free dyes have been washed away along with the filtered solution.
Qualification of materials
Result and Discussion
- Inter-Domain dynamics of native protein
- linker contributes freeier inter-domain dynamics
- Urea denature less compact region, showing naked dynamics
- pintide, barrier of the inter-domain dynamics
I26A is known to be a conservative mutant, so it can be used to understand some dynamics on proteins. There are some families of proteins that have similar or very similar characteristics, isoleucine and alanine can be one of the examples used for this experiment. When you look at FRET, in general, of the 98C and 126C with the FRET ratio of the I26A mutant at low FRET and high FRET ratio, it is more likely to be condensed at low FRET slope.
Alanine and isoleucine are grouped into the same category, being aliphatic with a hydrophobic side chain - the only difference being the carbon chain they have. It may be a very dangerous tool to use when injected excessively, but in other words, it can be used as a method to understand and observe which link or interaction of a particular mechanism is weakened, i.e. the native or favorable dynamics of the molecule. In many cases with macromolecules urea concentration up to 8 M has been used, but here we used a relatively very low concentration of urea that still shows very significant changes in the FRET tendency.
The higher concentration of urea, 0.1 M to 0.3 M urea, the higher the FRET efficiency was detected, while from 0.5 M urea concentration, it appears that the FRET efficiency is vaguely merged (showing about 0.5 FRET efficiency) and is decreased with higher urea concentration. (a) and (b) are each 98C and 126C FRET to directly understand how the FRET trend is changing along with progressively higher urea concentration at pin1. This may be an evidence that the weaker FRET, the further distanced interaction - mainly from the non-sustainable high FRET trend, but from the dynamics of active change observed, is less favorable, when the higher FRET trend and stable has strong position on this pin1.(8). pintide, interdomain dynamics barrier.
Pintide, a substrate known to interact with pin1, has inhibitor-like activity on the protein of interest. Pintide is a ligand mimic that competes for the binding of the natural ligand to the WW-domain of Pin1. Similarly, binding interactions between the WW-domain of Pin1 and phosphorylated ligands can be enhanced by phosphorylating specific amino acid residues in the WW-domain and the targeting ligand.
Its dissociation value, Kd value is known as 50uM, which indicates that at 50uM concentration of pintide on pin1 environment, half of the substrate interacts with Pin1, according to the theoretical understanding. Therefore, we designed to make a concentration of pin1 much less and much more than Kd value, 10uM and 100uM each. To also see the native dynamics of Pin1 with pintide substrate, we performed this experiment on vesicle design, provided with pin1 inter-domain FRET efficiency also on surface design.
With less concentration of pintide, 10uM environment, because very dramatically it is shown that there are huge losses in the high FRET efficiency area, while the low FRET ratio has increased extremely. This can be interpreted as strong evidence of pintide, and even as a competitive inhibitor for interdomain interaction of Pin1.
Conclusion
