Protein Purification and Analysis Day 4. Amino Acids, Peptides, and Proteins.

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

+NH3----------SGYAL-----------COO-

Peptides canionize

Peptides canbe charged

Proteins are a variety of sizes

Proteins can contain very

different ratiosof amino acids

1º 2º 3º 4º

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

Hydrogen bond between(1) C=O===H-N (4)

Alpha Helix

Beta sheet structuresSide 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, 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

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 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).

Column Chromatography

Ion ExchangeChromatography

Size-exclusionChromatography

AffinityChromatography

SDS binds to protein molecules. One SDS per two amino acids

SDS-PolyAcrylamide Gel Electrophoresis(SDS-PAGE)

SDS-PAGE of aprotein mixture

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.

Isoelectric focusing (IEF)

2-D electrophoresis

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