Bistable Isoelectric Point Photoswitching in Green Fluorescent Proteins Observed by Dynamic Immunoprobed Isoelectric Focusing

Abstract

We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the E222G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF were employed to monitor dark (non-fluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3–4 formal charges distinguish the bright and dark populations of avGFP, compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen bond clusters within the GFP β-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events.

Detection of Isoforms Differing by a Single Charge Unit in Individual Cells

“…IEF has immense resolving power and selectivity; even single-charge differences among proteoforms are detectable. [17]…”

“…The tGFP isoforms arise from differential C-terminal cleavage by non-specific proteases[17] and differ by just a single charge unit. Interestingly, the native conditions yielded 86% higher total tGFP probing signal than denaturing conditions, attributed to the sensitivity of photocapture efficiency on protein state or, possibly, to incomplete electromigration out of the microwell, as is under study (Figure 3c).…”

Microfluidics: reframing biological enquiry

“…Microfluidic isoelectric focusing, which separates biomolecules on the basis of charge differences, identified charge-switching in the fluorescent protein GFP upon stimulation with light 85 . Real-time imaging of the ~1-second microfluidic separations was crucial for observation of the charge switching and for determining switching kinetics.…”

“…Microfluidic tools have precisely defined features and contain highly complex channel networks and fluidic components that can execute specific functions 13,48,91,140–144 . In the past decade, the increasing availability of fluidic components and number of experienced microfluidic researchers have opened the door to new biological applications; these include new single-cell measurements 5,12,21 , high-throughput monoclonal antibody screening 6 , analysis of dynamic phenomena 21,77,85 and organ-on-a-chip platforms 15 . PDMS, polydimethylsiloxane.…”

Streptomyces violaceoruber Phospholipase A2: Expression in Pichia pastoris, Properties, and Application in Oil Degumming

“…Isoelectric point (pI) was determined according to the method of isoelectric focusing by Hughes et al [29]. Isoelectric focusing was traditionally carried out in gels and, recently, more in capillaries [30].…”