I performed deletion mutagenesis along the c terminal region of a protein targeting two specific leucine residues, and showed no change compared to wild type, as assayed by dual luciferase-renilla. However, when the leucine residues are substituted with alanine, there is a significant change. Alanine is smaller non-polar amino acid compared to leucine, but I'm having difficulty understanding why this would yield a significant result compared to knocking out the residues all together...
Additionally, these residues are on a C-terminal alpha-helix, followed by a region of intrinsic disorder, before protein end.
Hi there,
What are the residues immediatly downstream the 2 Leu? Are they similar (ie. bulky hydrophobic residue)? In this case deletion might not be deleterious because the immediate neighbouring residue is able to play the same role as the Leu within the structure. Can you modelize the helix and the impact of deletion on its overall structure? In case of alanine, methyl side chain would be too short to efficiently replace Leu.... BTW what do you mean by "significant change" about the ala mutants? Is it about expression/stability of the protein or about it's function?
I think that it is probably due to conformational or folding modification
Amended on 11 February 2018 (Today, it is said that Japan has been established for 2678 years ago)
I am pleased to hear that Leu and Ala exchange changes the enzyme ability or activity. I have previously observed that the hydrophobicity (Hyd) is the very important factor in Lipoamidase (please see file; Multiple hydrolase LIP). Therefore, we have published about the effect of surfactants on the biotinidase activity (please see file; Surfactant BIN), and the effect of phospholipids on the lipoamidase activity (please see file; Phospholipids LIP).
I am now considering that glyco-chain structure is more important in human diseases via the enzyme characters such as Km and heat stability of enzyme kinetic parameters (please see file; Median and Range of Biotin and JMBT Alopecia).
By the way, we have found that sufficient hydrophobicity (Hyd) is very important for the purification of hydrophobic membrane proteins. Thus, protein (including membrane proteins) determination by HPLC-SEC method has only been succeeded by using Brij 58 (polyethyleneglycol 1000 monocetylether) (please see file; HPLC-Surf-SEC protein determination method). Brij 58 contains the hexadecanoyl (cetyl, palmitoyl) residue. SDS, Nonidet P-40, Tween 20, and Triton X-100 (possessing shorter fatty-acyl chains than palmtoyl chain) have all given unsuccessful results; i.e., the column inlet-pressure gradually increases during human bile analysis (our unpublished observation).
We know that if any mutation either deletion of mere substitution affects the structural integrity of a protein, there is a very high tendency the protein looses its functionality unless the binding and active sites are preserved. Interestingly, the substitution of Leu by Ala residues could have a profound effect on the protein if the preceding or succeeding amino acid to the Leu residues do not cushion that effect of R-group on the helical structure.
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