Question

1.Ansawer the following questions about chemical synthesis of Peptides: A. What groups must be blocked in chemical synthesis of peptide? B. In what order are the amino acidss added? C. Described Step-by-step how to chemically synthesize a dipeptide.    2. what are the two main methods used to inhibt proteases after cell lysis? 3. List three ways proteins can be denatured once a cell is broken open. 4. Explain the journey of mRNA molecule through the secretory pathway.

Answer 1

1A. The amino acid N-termini are protected(blocked) by groups that are termed "temporary" protecting groups. Two common N-terminal protecting groups are tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc).

1B.Chemical peptide synthesis most commonly starts at the carboxyl end of the peptide (C-terminus) and proceeds toward the amino-terminus (N-terminus). ( Note : Protein biosynthesis (long peptides) in living organisms occurs in the opposite direction).

1C.The classical approaches to peptides production are called liquid-phase peptide synthesis and solid-phase peptide synthesis (SPPS). These two methods can be combined in a process called native chemical ligation. LifeTein’s standard peptide synthesis process involves the solid phase. The liquid-phase approach is used for the synthesis of short peptides, such as di- and tripeptides, and C-terminally modified peptides, such as enzyme substrates.

The controlled peptide synthesis requires selective protection and deprotection of the various functional groups: the amino group, the -carboxyl group or the side chain functional groups. The side group gives each amino acid its distinctive properties and helps to dictate the folding of the protein.

Principles of Solid-phase Peptide Synthesis

SPPS involves repeated cycles of coupling, washing, deprotection, and washing. A single cycle consists of the following:

• Cleavage of the alpha-amino protecting group
• Washing to remove the cleavage reagent
• Coupling of the protected amino acid
• Washing to remove excess material

Unlike ribosome protein synthesis, artificial synthesis builds peptides in the C to N direction. During solid-phase peptide synthesis, each peptide is anchored to an insoluble polymer at the C-terminus.

The free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which another amino acid may be attached.

Then the peptide is assembled by the successive addition of protected amino acids.

Once synthesis is complete, the desired peptide is cleaved from the resin. Usually, this cleavage step is performed with acids of varying strength.

The by-products can be removed by repetitive washings with appropriate solvents.

2. Protease inhibitors are molecules that block the activity of proteases, and typically function on classes of proteases with similar mechanisms of action. They are

Reversible inhibitors : Competitive, Uncompetitive and Non competitive

Irreversible inhibitors

suicide inhibitors.

3. Some cell lysis and protein solubilization methods cause the denaturation of proteins.Detergents are a class of molecules whose unique properties enable manipulation (disruption or formation) of hydrophobic–hydrophilic interactions

detergents have hydrophobic-associating properties as a result of their nonpolar tail groups. Nevertheless, detergents are themselves water-soluble. Consequently, detergent molecules allow the dispersion (miscibility) of water-insoluble, hydrophobic compounds into aqueous media, including the extraction and solubilization of membrane proteins.

Detergents at low concentration in aqueous solution form a monolayer at the air–liquid interface. At higher concentrations, detergent monomers aggregate into structures called micelles. A micelle is a thermodynamically stable colloidal aggregate of detergent monomers wherein the nonpolar ends are sequestered inward, avoiding exposure to water, and the polar ends are oriented outward in contact with the water.

4.The endoplasmic reticulum is the first step in the secretory pathway. Its membrane is continuous with the outer nuclear membrane. mRNAs drift around in the cytoplasm until they get picked up by a ribosome interested in translating them.

In ‘posttranslational translocation’ the new protein is moved into the ER after it’s translated. In the more interesting phenomenon called ‘cotranslational translocation’ the ribosome starts translation just like any other protein.