Hydrophobicity, Desolvation Energy, and weakness of method(s)
hydrophobicity: protein core buries hydrophobic residues.
desolvation energy: the desolvation energy of amino acids is important to the stability of the protein
- could cost energy if the peptide bonds don't reform the HBs when refolding. This debt is occurred by the desolvated polar groups which could destabilize the protein
Method 1: dG= -RTlnKeq w/ Keq= [apolar]/[water]
- the measure of a molecule going from water to apolar. the desolvation energy is the dG of the transfer. w/ this transfer, if dG<0, then hydrophobic.
- not good method bc of the fact that the transfer would be different for the different apolar solvents and there would be no definite way to classify the different amino acids in each apolar solvent.
Method 2: Clathrate method
- measures the volume of a structure/molecule and how much energy it takes to make a small hole in the water. No real flaws in this method.
- the size of the clathrate is directly proportional to the hydrophobicity.
- hydrophobicity (DE) is proportional to the increase in surface area of the ordered water
hydrophobic molecule has dG= 11.4kj/mol of apolar--> water ([aq]/[apolar]) @ RT. If [hydro molecule]=10mM in H2O, what is [apolar]?
dG=-RTlnKeq
11.4= -5.7 lnKeq
-2= lnKeq
Keq = 1/100
10mM= .01M ------> 100* .01= 1M of hydrophobic molecule in apolar solvent
Gly and Pro special?
Glycine is the only achiral amino acid.
Proline is imino acid and has alpha C and imino group connected in 5 member ring "pyrollidine"
Amino acid useful for calculating molar concentrations of protein?
tryptophan and its 280nm UV absorbance. found in a lot of peptides/proteins
- phenylalanine and tyrosine could also be used similarly but the main contributor is the tryptophan
amino acid participate in redox reactions and form crosslinks
cysteine and forms crosslinks from the sulfur groups.
BME=beta mercaptoethanol which is used to reduce disulfide bonds
The disulfide bond reaction bw 2 cystines ---> knowing the direction of the oxidation and reduction
glutathione
tripeptide (ECG-Glutamate-cysteine-glycine) w/ a reducing thiol (in cysteine) that reducing conditions in the cell.
memorize the structure and pka of the 7 titratable amino acids
DECHKYR -- 1 letter abbrev.
pI of protein; what functional groups or amino acids contribute to the pI of a protien?
pI is the isoelectrical point of the titration curve; it is where the amino acid has no overall net charge.
NH3+, COO-, and the R groups contribute to the pI of the proteins
A (pI=6) and B (pI=7). Which is less positive/more negative at pH 4? What are their charges at pH = 6.5?
@ pH4 they are both protonated, hwr, A is more negative than B. @ pH 6.5, A is deprotonated (negative charge) while B is still protonated (positive charge).
----best to draw out a pH vs charge plot
anion and cation exchange? A w/ pI=7 and B w/ pI=5, strategy for separating 2 using ion exchange chromatography.Charge of A @ pH = 5,7, 8?
--- pH and resin that both proteins will bind, which is eluted first?
anion -binds anions while cation- binds cations
---- pH 6 with anion to separate them.
A @ 5= positive, 7= neutral, 8= negative
both bind at anion 8 with A eluting first
affinity chromatography? pH affect hexahistidine tag?
affinity chromatography-- affinity resin, ligand, and protein. ligand= genetically encoded tag inserted into protein sequence. affinity and ligand + protein fit together in the column.
His-tag attaches when it is deprotonated, therefore it doesn't bind when it is protonated. therefore it will only bind w/ pH larger than 6 because pka = 6.1 (memorized value)
Fred Sander sequencing insulin?
implies template in cells used to make proteins---the same protein over and over again w/o fail. ---foreshadowed idea of codons and how DNA works
primary structure= sequence of amino acids beginning w/ amino terminus and ending w/ c-terminus.
protein= single chemical substance
sanger's work came out before cricks' would predicted that codons would be 3 nulceotides long and would code for amino acids
Assume His in a serum protein has a pKa of 7.4, and it is in position to form a salt bridge with a nearby Asp. Predict whether the salt bridge will form or break at a given pH. Is the salt bridge stronger at pH 7.0 or 6.0? Does the salt bridge form at pH 8.4, or is it simply much weaker? HINT: what is the ratio of deprotonated: protonated His at pH 8.4?
7.4= protonated HIS(10.1) and deprotonated ASP (4.1), so they could form a salt bridge.
stronger at 6 or 7 pH: stronger at 6 because His is more positive and less likely to be a deprotonated His.
---think of it as distance from pKa. pH 7: more neg. asp, but less pos. his== you want as close to equal as possible.
The protein-folding problem (for soluble proteins) is based on four basic principles. What are they?
1. bury hydrophobic groups
2. satisfy polar and charged groups
3. minimize surface exposure by packing atoms densely
4. obey stereochemistry restraints
2 problems in protein folding did Pauling's Alpha helix and beta sheets correctly address years before protein structure was solved.
---- need for H bonds to be statsified
---- peptide plane restriant being obeyed
Earlier, you learned that H-bonds and Salt-bridges are strong, but they contribute little to the stability of a folded protein. Why? If folded protein contains an ‘unsatisfied’ H-bond donor in the hydrophobic core, why is that destabilizing?
all the H-bonds and salt bridges have to be satisfied in the folded structure for the energy cost to be paid back. The misfolded state, even if it is partially misfolded, will degrade back because it is at a higher energy state. Specifically, in the hydrophobic core, an unsatisfied H-bond went from a state where it was interacting with water (unfolded ---> H-bonds) to a state where there are no interactions which would cost energy