Biochem - Lecture 3 pt 4

Terms	Definitions
Quaternary structure describes the interaction...	of individual polypeptides already folded in their globular form
The same factors that influence teriary structure will affect quaternary as well:	need to bury hydrophobic groups, and ionic, H-bonding, disulfide formation, and metal chelation - shape is very important.
Polypeptides found in a multi-chain protein are usually called	subunits
A wide variety of quaternary structures are known -	some large proteins have more than 10 different subunits.
Hemoglobin is a classic example of a	multisubunit protein, consisting of two α- and two β-subunits, which interact closely through complementary shapes, hydrophobic surfaces, ionic interactions, and H-bonds.
Protein folding is a ordered process that seems to follow a series of steps that are unique to each protein	The process is dependant on the AA sequence
Predicting the steps in folding anything but small peptides still...	defies even the fastest computers
In some cases, molecular chaperones (proteins that bind partly folded polypeptides) assist in folding...	they seem to stablize key intermediates thereby preventing non-specific aggregation and incorrect folding.
Despite the unique folding pathways...	a limited number of folding motifs have been recognized
Protein denaturation is	the process by which a folded or native protein is converted to an unfolded form
The free energy difference between the native and unfolded states of a protein is	usually small (Delta G folding~ 5-10 kcal/mol), so mild treatments like heat or a change in solvent will denature most proteins
Once denatured....	a few proteins can refold spontaneously but this is rare ( chaperones are usually required)
Most denatured proteins....	tend to aggregate and precipate (like the proteins of egg white)
When egg white is heated, the proteins denature:	hydrophobic R-groups are exposed to the H2O and the proteins aggregate and become insolube.
The most familiar denaturants are:	- Heat (T> 55 degrees for most proteins) - Ph (disrupts H-bonds and Ionic bonds) - organic solvents
High concentrations of certain solutes which disrupt the H-bonding system of water (8M urea, 6M guanidine hydrochloride) are...	excellent protein denaturants, and keep the denatured form in solution
Proteins, besides water, are the most abundant substance in...	most cells. 10 - 20% by weight.
Protein Functions with examples: - Catalysis	Enzymes
Protein Functions with examples: - Transport	Hemoglobin
Protein Functions with examples: Structure	Collagen
Protein Functions with examples: Contractile	Actin & Myosin
Protein Functions with examples: Nutrient	Ovalbumin
Protein Functions with examples: Defense	Immunoglobins
Protein Functions with examples: Regulatory	Insulin
Humans manufacture at least 25, 000 different proteins
Some proteins are simple polypeptides, or are...	- conjugated with sugars (Glycoproteins) - with lipids (lipoproteins)
Others require non-amino acid..	cofactors or prosthetic groups for full activity
Cofactors may be.. and they maybe covalent/non-covalently attached to the protein	- inorganic e.g metal ions - organic e.g sugar, lipid, heme, flavin
enzyme cofactors are called...	coenzymes
Proteins may be purified on the basis of differences in:	1) size 2) charge 3) solubility 4) affinity for materials
Ion exchange chromatography can be used to...	seperate proteins with different pIs.
Size exclusion or Gel sieving Chromatography	Small beads of polymerized glucose, agarose, or acrylamide are manufactured with different sizes of poe depending on the degree of cross-linking of the polymer.
The beads are packed into...	..a clyinder and a mixture of proteins is applied.
Big proteins don't...	enter the porous beads, and run quickly through the column
Small proteins enter and exist the beads and..	elute more slowly
Ion-Exchange Chromatography	Polystyrene or silica-based beads have anionic or cationic functional groups attached.
Sulfonic acid is a strong acid and is...	anionic at pH 2, whereas all amino acids are cationic.
Ion-Exchange to separate amino acids: 1) Beads with sulfonic acid groups attached are...	... packed into a cylindrical column
2) A mixture of AA is applied in a...	... buffer at pH 2 and bind to the sulfonic acid groups
3) The AA are removed by...	washing the beads with buffers at higher pH
4) When the pH reaches the pI of an AA..	it binds from the beads and washes out of the column
5) Differences in the pI of each AA cause them to be...	... dissociated at slightly different pHs
6) Adding salt will also remove the AA by...	...competing with the AA for the binding sites
example of an Ion exchange chromatography
The same ion-exchange approach can be used to seperate and purify proteins because...	all proteins carry a mixutre of postive and negative charges. They have weak acid/base character of ionizing side chains (Asp, Glu, Lys, His, Arg, Cys, Tyr)
The state of protonation of each group depends on its pKa...	so the net charge on a protein is +ve at low pH and becomes more -ve as pH is raised
At a certain pH,	-ve and +ve groups are equal and we have the pI. Different proteins will have different pIs
A ligand is any molecule that is...	bound specifically by a protein e.g ATP binds to hexokinase
Affinity Chromatography 1.	Ligands are attached to polymer beads
Affinity Chromatography 2.	A mixture of proteins is applied to the beads
Affinity Chromatography 3.	Hexokinase binds, all others are washed out
Affinity Chromatography 4.	ATP is added and competes for the binding site causing pure protein to unbind and elute from the column.
SDS-PAGE	Gel electrophoresis is an analytical technique used to estimate the mass of a protein
SDS-PAGE 1)	The gel is a cross-linked polyacrylamide molecular sieve
2)	The detergent sodium dodecyl sulfate (SDS) binds to the proteins. About 1 SDS per 2 AA.
3)	SDS-coated proteins move through the gel when an electric potential is applied
4)	After electrophoresis the proteins are visualized by staining with Coomassie Blue or Silver
5) The migration distance is proportional to the ...	..log10 [Mr]. The mass of an unknown protein can be determined by interpolation using the migration distances of proteins of known molecular weight.
Example of SDS-PAGE