Protein charge 101
Define the iso-electric point (IEP)
What is the point at which a protein has a zero net charge?
Protein netto charge at a pH lower/higher than its IEP
What is the isoelectric point, where the net charge is zero; below it, proteins gain protons and become positive, above it, they lose protons and become negative?
The iso-electric point of GFP
What is 5.5?
Fluorescence working technique
What is a molecule absorbs light, loses some energy as heat, and re-emits light at a lower energy (different color)?
pH at which GFP is soluble and fluorescent and why
What is GFP has an isoelectric point of 5.5, and is most soluble and fluorescent at a pH between about 6.5 and 9.0 due to solubility being best at a pH away from it's IEP?
Amino acids that can be charged in a protein
What is amino acids containing ionizable side chains or terminal groups?
Amino acid present at which pH (low or high) determines the charge
What is amino acids in high pH have negative charge, in low pH have positive charge?
pH in which GFP is badly or very well soluble
What is negative charge of GFP in higher pH; positive charge of GFP in lower pH?
Effect of pH on fluorescence
What is low pH reduces fluorescence by protonating the chromophore, since its fluorescence depends on its protonation state?
Variants of GFP that are more soluble and fluorescent, if the solubility and fluorescence of GFP in soft drinks are not favorable
What is Superfolder GFP (more soluble and acid-resistant due to extra folds) and Enhanced GFP (brighter, more soluble, and also acid-tolerant)?
The charge of amino acids in a protein (negative/positive?)
What is D, E, Y and C are negatively charged; H, K and R are positively charged?
The relationship between charge and solubility of a protein
What is a protein is more soluble when highly charged and less soluble near its isoelectric point?
GFP solubility in soft drinks
What is GFP remains soluble in soft drinks because their low pH is far below its isoelectric point, giving it a net positive charge and reducing aggregation?
Structure (chromophore) responsible for the fluorescence of GFP
What is the fluorescence of GFP comes from an internal chromophore, formed post-translationally from Ser65-Tyr66-Gly67 by cyclization and oxidation?
Addition of alcohol influence on solubility, fluorescence and pH of of GFP
What is alcohol lowers solvent polarity, reducing protein solubility and causing aggregation or precipitation, while partially denaturing the protein and harming its fluorescence?
Determination of IEP
What is identifying the ionizable groups (including N-terminus, C-terminus), determining the net charge at different pH values and calculating the pH when the charge net is 0?
The consequence of the relationship between solubility and IEP
What is the further the pH is from a protein’s isoelectric point, the more soluble it becomes?
Predicted effect of alcohol on the solubility of GFP
What is alcohol reduces GFP solubility by lowering solution polarity, disrupting hydrogen bonding, and possibly denaturing the protein?
Wavelengths for excitation and emission of different GFP proteins
What are fluorescent protein classes and wavelengths:
Class 2 (enhanced GFP) excites at 488 nm and emits at 509 nm;
Class 3 (Topaz) excites at 514–528 nm and emits at 540–550 nm;
Class 4 (YFP) excites at 514 nm and emits at 527–530 nm;
Class 5 (CFP) excites at 433–450 nm and emits at 475–485 nm?