1. Amino acid substitution & Free Reducing free sulfhydryl residues
PADMABALAJI.P
ll M.S.c., Marine Biotechnology

2. Definition for amino acid substitution
The naturally occurring or experimentally induced replacement of one or more amino acids in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties.

3. Continuation:
Sometimes proteins losses activity due to changes in amino acid.
Amino acids are substituted to enhance their activity.
Eg: aspargine & glutamine is converted to isoleucine and threonine

4. Protein undergo deamination at high temperature.
Releases ammonia,loss of amide moiety
Aspargine & glutamine Asparticacid+glutamic acid
Loss of protien activity

5. Changing in yeast
Aspargine in position 14 & 78 are changed by oligonucleotide directly mutagenesis to theronine or isoleucine
This enhanced the thermostability.

6. Stability at 100oC of yeast enzyme`
a.a at position 14
a.a at position 78
Half life
(min)
Wild type
Asn
Asn 13
Variant
Asn
Thr 17
Variant
Asn
Ile 16
Variant
Thr
Ile 25
Variant
Asp
Asn 11

7. Basic facts about SNPs
SNPs occur every bases in human genome;
Two of every three SNPs involve the replacement of cytosine (C) with thymine (T);
SNPs can occur in both coding (gene) and noncoding regions of the genome;
Many SNPs have no effect on cell function, but others could predispose people to disease or influence their response to a drug.

8. Basic facts about SNPs
SNPs occur every bases in human genome;
Two of every three SNPs involve the replacement of cytosine (C) with thymine (T);
SNPs can occur in both coding (gene) and noncoding regions of the genome;
Many SNPs have no effect on cell function, but others could predispose people to disease or influence their response to a drug.

9. Base-Pair Substitution Ex) Missense Mutation
One Amino Acid Substituted for Another
Sickle Cell Anemia
Valine is replaced with Glutamic Acid

11. Free sulfhydryl residues
Protein engineering sometimes results in less activity of protein than expected.
Formation of dimers & higher oligomers which are inactive.

12. Analysis of DNA sequence confirms
Some proteins consist of more cystein residues
Resulted in intermolecular disulphide bonding
Converting cystein to serine can diminish these dimers,

13. Cont.. Why select serine? Cystein & serine has same structure
Cystein-SH, serine –oxygen
Primary structure of protein analyzed to find position of cystein
Protein now stable & native form

14. Thank you