Chemical Modifications of Proteins Flashcards
What are some reasons to chemically modify proteins?
- To provide mechanistic or structural information
- Indicate highly reactive groups
- Measure exposure of residues
- Provide useful alterations of structure
- Introduce spectoscopic probes or radioactive tags
- Allow for cross-linking of proteins
What are three experimental considerations when making chemical modifications to proteins?
- Extent of modification : amount of reagent, pH, presence of denaturants. Can lead to all molecules being partially modified or molecules modified to different extents.
- Specificity and side reactions : most reagents will react with multiple types of residues, thus the effects of specific residues should probably be done more with precise genetic engineering. Active site residue might be very reactive.
- Changes in conformation : The protein may denature when attempting to modify it either due to the actual chemical reaction or the specific chemical modificaiton. Thus it must be determined if conformation was what caused a change in activity.
The reactive amino acids tend to be nucleophiles (give a list) while the modification reagents are generally electrophilic.
Cys, Met, Arg, Lys, His, Asp, Glu, Trp, Tyr
What are three reducing agents for disulfide bonds?
- 2-mercaptoethanol (BME)
- Dithiothreitol (DTT)
- Tris(2-carboxyethyl)phosphine (TCEP)
How can you purify a protein? (step by step)
-lyse E.coli with: buffer at pH=7.4, salt, low imidazole, glycerol, protease inhibitor
- Centrifuge and collect soluble fraction
- IMAC, bind, wash, and collect elution. Elute with imidazole gradient add EDTA and reducing agent
- Dialysis with buffer at pH =7.0
- IEC. (specific cation exchange). Elute with high salt gradient.
- SEC and concentrate protein to 1-2mL or HIC and dialyze again.
5,5’-dithiobis (2-nitrobenzoate) (DTNB)
It is used to quantify free cys residues by measuring the production of nitrothiobenzoate dianion which has a strong absorbance in visible light.
At what pH are Iodoactate and Iodoacetamide specific for Cys alkylation?
pH of 6.
Give an application of Iodoacteamide
stopping the reaction of a cys protease like TEV or papain.
What are three functions of cross-linking within polypeptides
- Determine if two polypeptides are near each other, or if two amino acid residues are near each other.
- Stabilizes tertiary structure to make protein more rigid and stable.
What are properties of linker cross-linking reagents?
- Permanent
- Cleavable
- Length determines the max distance between cross-linked groups.
What are three reasons for cross-linking polypeptides to other molecules?
- Identify protein-protein interactions (subunits in oligomers or transient interactions)
- Measure distance between polypeptides (Lys-specific homobifunctional reagents used since Lys is often found on protein surface)
- Attach protein to a carrier (affinity columns or conjugate vaccines)
Name three cross-linking reagents:
- Glutaraldehyde
- 3,3’-dithiobis (DTSSP)
- APDP
How can DTSSP be used to identify protein complexes?
- React DTSSP with protein mixture. Separate cross-linked proteins by molecular weight in first dimension
- cleave cross-linker in gel with disulfide reducing agent.
- Separate proteins by molecular weight in the second dimension
- Identify proteins in the complex
How can APDP be used to identify protein-protein interactions?
- react I-APDP in dark with protein of interest
- Mix with other proteins to reconstitute the biological complex
- Illuminate cross-links to nearest neighbors
- cleave cross-links with reducing agents
- Identify the partner of the protein of interest with the radiolabeled Iodine still present by polyacrylamide gel electrophoresis.
