protein purification and analysis day 4. amino acids, peptides, and proteins
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Polypeptide chains fold upon themselves to form aunique three-dimensional structure.
There are four defined levels of protein structure
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 ofatoms. Proteins in their functional, folded conformations arein 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
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, PheCharged amino acids are on the surface.
Arg, Lys, His, Asp, GluUncharged 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
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 concentrationsThis 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).
Native PAGE"Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS. While in SDS-PAGE 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.
Laboratory ExperimentGoals 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
H3N CH C
CH2
O
O
N
NH
N
NH