protein solubility and stability Flashcards
(28 cards)
factors that affect protein stability
-pH and temperature
-constituents like salts, organic solvents
-amino acid sequence and composition, 3D structures, exposure of residues, intramolecular interactions, protonation status
-solvent properties like polarity, bond interactions, density, forming abilities
in effect of pH, what is the effect when pH>pI, =pI and <pI
pH>pI
repulsion, net negative chare
=pI
aggregation, neutral no net charge
<pI
repulsion, net positive charge
what is the most important determinant of solubility of a protein
proportion of surface that is composed of charged side chains
what is pI
isoelectric point, pH value which molecule carries no electrical charge
equation for pI
pI=(pKa1+pKa2)/2
how to calculate pI of oligopeptides
idk yet
when calculating pI, if side chain has no ionisable group/acidic/basic/other then pI is…
no ionisable group=average of NH3 and COOH pKa
acidic=average sidechain pKa with α-COOH pKa
basic=average sidechain pKa with α-NH3 pKa
other ionizable groups (tyr and cys)=average middle pKa and α-COOH pKa
ionic strength equation
I=1/2 (sum of Ci Zi^2)with limits 1 to
(notes if unsure)
Ci=conc of ith ion
Zi=charge of ith ion
what is salting in effect
when solubility increases in presence of salt bc the salt ions shield the proteins charged groups and reduces electrostatic repulsion between them making the protein more stbale and soluble
more salt=more solubility
what is the solubility of proteins determined by
hydration of charged and polar amino acids at protein molecule surface
at low ionic strength, protein molecule is surrounded by…
ionic atmosphere described by debye-huckel theory
what happens in the salting in effect
theres an excess of opposite charge ions to net charge of protein that are attracted to the immediate surrounding of protein molecule
screening by counterions decreases the electrostatic free energy of protein resulting in increased solubility
what is the salting out effect
when presence of high concentration of salt decreases solubility of protein, due to salt ions competing with the protein for water molecules leading to protein aggregating and precipitating out of solution
high salt conc=less solubility
salting in vs out effect
in
more salt=more solubility
out
high salt conc=less solubility
what happens in salting out effect
ions compete with protein for water molecules and decreases solvation for proteins, salt increases surface tension of water which increases energy of cavity formation
what is denaturation of a protein
unfolding, disruption of tertiary/secondary structure
primary driving force of protein unfolding
reducing free energy due to contact of nonpolar residues with water
-common to have hydrophobic residues on surface and hydrophilic residues buried in core
-protein interiors are tightly packed
-many peptide groups and side chains buried in native become exposed to solvent in denatured
-pockets of structure in denatured
where are hydrophobic/phillic residues on proteins
hydrophobic residues on surface
hydrophilic residues buried in core
what are buried water molecules and what do they do
water molecules inside protein interior
-fills internal cavities and interacts with polar amino acid side chains within protein core, contributes to stability and function
effect of protein unfolding
-as protein gets bigger, more residues buried inside
-larger increase in burial of charged groups
-proportion of surface that is composed of charged side chains is important determinant of protein solubility
-denatured proteins expose buried non polar amino acids, solubility of denatured proteins in water is lower
how do hydrophobic particles in water aggregate
hydrophobic particles break hydrogen bonds between water molecules, this is endothermic reaction bc heat is put into system to break the bonds
water molecule will form new H bonds aorund hydrophobic particle forming a clathrate cage, this makes system more ordered with decreased entropy
what is entropy a measure of
disorder
in ∆G= ∆H - T ∆S, what does a positive ∆G mean
mixing of hydrophobic particles and water is not spontaneous, -ve means it is
why do hydrophobic particles aggregate
when hydrophobic particles meet and interact enthalpy increases (∆H>0) bc some H bonds that form the clathrate cage will break
-breaking down a portion of the clathrate cage causes entropy to increase due to release of ordered water molecules to the bulk
-∆S is dominating so ∆G is negative meaning hydrophobic interactions are spontaneous
-system prefers to stay at lowest overall free energy level so protein particles aggregate to have the lowest overall free energy
