CHE 350 Lecture Notes - Lecture 3: Differential Centrifugation, Amine, 2-Mercaptoethanol

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Denaturation and Renaturation
Low conformational stabilities = easily denatured by altering w/ weak nonbonding forces
by:
o1) HEAT = abrupt change “melts” cooperatively
o2) pH = alters ionization state of R-groups, changes charges, changes H-bonds
o3) Detergents = reacts w/ nonpolar residues, interferes with hydrophobic interxn
o4) Chaotropic Agents = gaunidinium ion, urea  increases solubility of nonpolar
stuff in H2O and disrupts hydrophilic interxns
Many denatured proteins can be renatured (kind of)
oEX: RNase A (124 residue single chain protein)
1) Unfolded, disulfide bonds (4) reductively cleaved w/ urea containing 2-
mercaptoethanol
2) Dialyze urea, reductant
3) Expose solution to O2 at pH 8  oxidizes SH groups to reform disulfide
bonds
4) Yield = 100% enzymatically active
Proteins are Dynamic
Proteins are flexible and rapidly fluctuating
Large collection of rapidly interconverting conformations with equal = stabilities
o= flexibility
Many proteins have unfolded regions
oIntrinsically disordered proteins
oHas sequences w/ polar and charged aminos
oLacks bulky hydrophobic groups
oInvolved in signaling and regulation
oOrdered proteins = catalytic rxns, transport, structure
oUnfolded = bigger interface = less crowding
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Protein Folding
Proteins fold to their native conformations via directed pathways, not randomly
As a protein folds, stability sharply, free energy sharply  FOLDING IS A ONE WAY
PROCESS
oUnfolded = high entropy, high energy = disordered state = many conformations
oFolded = low entropy, low energy
oFalse energy minima = partially folded confs temporarily trapped until
overcoming free energy barrier = energy conf
Overcome by thermal activation (heat) or molecular chaperones!
Molecular Chaperones
SUPER IMPORTANT
oEssential proteins that bind to unfolded and partially folded polypeptide chains
to disrupt improper associations of exposed hydrophobic segments that would
lead to non-native folding, aggregation and precipitation
oMost require ATP
oMost are ATPases = enzymes catalyzing ATP +H2O  ADP + Pi
oATP binds protein (enzyme), ADP releases protein (enzyme)
oThey lift polypeptides out of false minima
Ex: heat shock proteins (Hsp)
Hsp 70 w/ Hsp 40
Trigger Factor (prokaryotic)
Chaperonins
Hsp 90
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Chapter 5 – Primary Structure
Polypeptide Diversity
Primary structure = amino acid sequence of polypeptide chains
Sequence of nucleotides in gene = order of aminos in polypep = proteins
20 amino acids available
oProteins of n residues = 20n sequences possible
Proteins usually contain 40 residues (at least)  anything lower is just a peptide (tiny
polypeps)
Multisubunit proteins = contain several identical or nonidentical chains called subunits
Size Range = Optimization of Biochemistry
1) 40 residues is the near minimum for a polypeptide chain to fold into a discrete and
stable shape that allows it to carry out a particular function
2) Polypeptides w/ >>1000 residues may approach the limits of efficiency of the
protein synthetic machinery. The longer the polypeptide, the longer its
corresponding mRNA, the higher the likelihood of errors during transcription +
translation
The 20 aminos don’t appear w/ equal frequencies in proteins
Most abundant = Leu, Ala, Gly, Val , Glu, Ser
Rarest = Trp, Cys, Met His
The characteristics of a protein depend more on its amino sequence than on its amino
composition
oThicken and kitchen
Protein Purification and Analysis
1) First step in isolation  get it out of the cell and into the solution via cell lysing
2) One a protein is removed from its natural environment, pH, T, other conditions MUST BE
CONTROLLED for protein stability
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Document Summary

Large collection of rapidly interconverting conformations with equal = stabilities: = flexibility. Proteins fold to their native conformations via directed pathways, not randomly. As a protein folds, stability sharply, free energy sharply folding is a one way. Process: unfolded = high entropy, high energy = disordered state = many conformations, folded = low entropy, low energy, false energy minima = partially folded confs temporarily trapped until overcoming free energy barrier = energy conf. Overcome by thermal activation (heat) or molecular chaperones! Primary structure = amino acid sequence of polypeptide chains. Sequence of nucleotides in gene = order of aminos in polypep = proteins. 20 amino acids available: proteins of n residues = 20n sequences possible. Proteins usually contain 40 residues (at least) anything lower is just a peptide (tiny polypeps) Multisubunit proteins = contain several identical or nonidentical chains called subunits. The longer the polypeptide, the longer its corresponding mrna, the higher the likelihood of errors during transcription + translation.

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