Altering protein structure and studying proteins

Post Translational modification

Chemical modification of a protein after translation extends range of function of the protein.

Covalent post transitional modification

Covalent attachment of biomedical groups such as acetyl,hydroxyl phosphate lipid or carbohydrate.

Cleavage post transitional modification

Proteases can cut proteins of specific sites to change activity or function

pH change

pH affects the balance between protonated and deprotonated form

Mutations

Primary sequence dictates 3D structure and function so a mutation to this changes function and structure of proteins.

Proteome

The proteome of an organism tissue or cell is a complete list of all proteins at a specific time or environment. This is continuously changing.

Identifying proteins: SDS polyacrylamide gel

Separates proteins based on size. Molecules within net charge can move through anything in an electrical field.

Identifying proteins: Mass spectrometry

An analytical technique used to determine the masses of peptide and their sequences. 1.Ion source 2.Mass analyzer 3. Detector. Once finished you can compare to human protein database.

Proteomic studies

Large scale studies of multiple protein systems analysis of entire cells and organisms. Utilize tandem Mass spectrometry for multiple proteins.

Purification

To get a single protein is difficult so we exploit the difference between various proteins to seperate them. 1. salting out 2. gell filtration 3. Ion ecxchange chromatography 4. Affinity chromatography

Salting out

Separates proteins based on solubility. Precipitation by salt works because protein precipitate at different salt concentrations.

Ion exchange chromatography

Separation based on size in molecular weight. Porous insoluble beads trap and impede smaller molecules so larger ien flow rapidly and exit first

Affinity chromatography( very affective)

Separates proteins base on protein binding affinity. Utilizes specific binding property of proteins.

Detection of protein s

we detect / measure using enzymatic assay and specific antibody for the protein

Enzymatic detection

Observing products of enzyme catalyzed reaction.

3-D Structure Nuclear Magnetic Resonance

Reveles 3D structure of proteins in solution. Limited to proteins< 30 kj. Nosey measures dstance between nonbonded atoms in angstroms.

X-RAY crystallography 3-D structre

You need crystals and amino acid sequence to determine electron density map.

3-D Structure Cryo EM

Signal to noise ratio is very small so you need thousands of images to detect protein structure. Since sample is in solution shows different conformations