Protein Purification and Analysis
Day 4
Amino Acids,
Peptides,
and Proteins
Absorption of ultraviolet light by
aromatic amino acids
Amino acids
are
“zwitterions”
at pH 7
Titration of
glutamate
Titration of
histidine
Formation of a peptide bond
Ser – Gly – Tyr – Ala - Leu
+NH
3
----------SGYAL-----------COO
-
Peptides can
ionize
Peptides can
be charged
Proteins are a variety of sizes
Proteins can
contain very
different ratios
of amino acids
Polypeptide chains fold upon themselves to form a
unique three-dimensional structure.
There are four defined levels of protein structure
1º
2º
3º
4º
1. The three-dimensional structure of a protein is determined by its
amino acid sequence.
2. The function of a protein depends on its structure.
3. Each protein has a unique structure.
Chymotrypsin
Proteins have a specific conformation, the spatial arrangement of
atoms. Proteins in their functional, folded conformations are
in their native state. Native conformations are not very stable.
Glycine
Question:
Is the structure of every protein totally
different, or are there common themes?
Answer:
There are regular folding patterns of the
peptide backbone present in most proteins.
Examples: alpha helix
beta sheet
Alpha Helix
Hydrogen bond between
(1) C=O===H-N (4)
Beta sheet structures
Side chains of adjacent amino acids protrude in opposite directions
Beta sheet structures
Many regions of secondary structure are connected by b turns
Tertiary (3º) structure of proteins
Proteins fold into a globular structure that
excludes H2O from the interior.
There is a systematic arrangement of amino
acid side chains in proteins.
In general:
Nonpolar amino acids are in the interior.
Val, Leu, Ile, Met, Phe
Charged amino acids are on the surface.
Arg, Lys, His, Asp, Glu
Uncharged polar amino acids are on the surface
or in the interior.
Ser, Thr, Asn, Gln, Tyr, Trp
Quaternary (4º) structure of proteins
Some proteins form aggregates of 2 or more subunits.
Reasons for multiple subunits in a protein include:
1. Cooperativity between subunits of a protein may be an important part of a
protein’s function. Example: hemoglobin binds oxygen cooperatively.
2. A protein’s catalytic function may require amino acids from each subunit.
Example: active HMG-CoA reductase is a dimer.
3. To fulfill a specific function a protein may have to be too large to be
synthesized as a single subunit.
Example: groEL chaperonin has 14 subunits
Working With Proteins
Experimental techniques for
protein analysis
and characterization
A cell contains many types of proteins
In the lab we want to isolate a single protein for experiments
We first grow cells or isolate tissues that contain the protein of interest
We break open the cells (lysis) to produce a crude extract
Use centrifugation to separate soluble from insoluble material
We fractionate the protein mixture based on properties of
individual proteins such as size, charge or solubility.
By using a combination of fractionation procedures a protein can be
isolated from all other contaminating proteins in a cell.
This process is called protein purification.
Fractionation by relative solubility
The procedure of ammonium sulfate (AS) precipitation is used to
separate proteins on the basis of their relative solubilities.
The solubility of proteins is lowered at higher salt concentrations
This is called salting out.
As the amount of AS is increased, more proteins precipitate.
A protein chemist wants to determine where the protein of interest
precipitates and other proteins do not (or vice-versa).
Column Chromatography
Ion Exchange
Chromatography
Size-exclusion
Chromatography
Affinity
Chromatography
SDS binds to protein molecules. One SDS per two amino acids
SDS-PolyAcrylamide Gel Electrophoresis
(SDS-PAGE)
SDS-PAGE of a
protein mixture
Native PAGE
"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDSPAGE the electrophoretic mobility of proteins depends primarily on their molecular mass, in
native PAGE the mobility depends on both the protein's charge and its hydrodynamic size.
The electric charge driving the electrophoresis is governed by the intrinsic charge on the protein
at the pH of the running buffer. This charge will, of course, depend on the amino acid
composition of the protein as well as post-translational modifications such as addition of sialic
acids.
Since the protein retains its folded conformation, its hydrodynamic size and mobility on the gel
will also vary with the nature of this conformation (higher mobility for more compact
conformations, lower for larger structures like oligomers). If native PAGE is carried out near
neutral pH to avoid acid or alkaline denaturation, then it can be used to study conformation,
self-association or aggregation, and the binding of other proteins or compounds.
Native gels can be sensitive to a process that alters either the charge or the conformation of a
protein, such as protein-protein or protein-ligand interactions
It is possible to recover proteins in their native, active state.
Isoelectric focusing (IEF)
2-D electrophoresis
Laboratory Experiment
Goals of Lab: Purify a recombinant form of the enzyme catalase expressed in the
bacterium Escherichia coli (E. coli). The enzyme produced is the form of catalase found
naturally in the bacterium Listeria monocytogenes. The gene encoding L. monocytogenes
catalase gene was fused to a circular plasmid DNA used to produce proteins in E. coli.
•
IMAC (immobilized metal affinity
chromatography) will be used for
purification
Histidine
Imidazole