harges on amino acids and proteins Acidic side chains: glutamate and aspartate A A- + + + - + Basic side chains: arginine, lysine & histidine Glycine @ p 1 B+ B + + + The amino group, pka 9.6 3 N+ The carboxyl group, +N 3 N 2 + verall charge +1 Glycine @ the pka of the carboxyl group, p 2.2 Glycine @ p 7 50% in the Acid form, A 3 N+ 50% in the conjugate Base form, A- 3 N+ - The amino group, +1 3 N+ The carboxyl Group, -1 - verall charge, 0 1
Glycine @ the pka of the amino group, p 9.6 Glycine @ p 12 50% in the acid form 50% in the conjugate base form 3 N+ - 2 N - 2 N - verall charge, -1 The pi The pi is the p where the charge on the amino acid is exactly zero. If the amino acid is placed in an electric field at this p it will not move. If you place the amino acid in a solution at a higher p it will be negative. If the solution has a lower p the amino acid will be positive. alculating the pi of Glycine Find the range of ps where the overall charge of the majority of molecules is zero In the case of glycine this is between the pkas 2.2 and 9.6. Find the average = 5.9 p [-] [-] p 1 p 7 p 14 [-] Increasing acidic = [-] Increasing [-] basic [-] +1 3 N+ 2
[-] pka 9.6 [-] [-] pka 9.6 [-] +1 0 +1 0-1 3 N+ 3 N+ - 3 N+ - 2 N - pka 9.6 [-] pi 5.9 pka 9.6 [-] [-] pi 5.9 [-] Glycine will be positive (+) Glycine will be negative (-) +1 0-1 3 N+ - 2 N - 3 N+ 2N - pka 9.6 [-] pi 5.9 [-] [-] [-] + - +2 +N 3 3
pka 9.0 [-] [-] pka 9.0 pka 10.5 [-] [-] +2 +1 +2 +1 0 - - 2N - pka 9.0 +N 3 +N 3 +N 3 +N 3 +N 3 pka 9.0 pka 10.5 [-] [-] pka 9.0 pka 10.5 [-] [-] pi 9.8 +2 +1 0-1 verall harge 0-2N - 2N - - 2N - 2N - pka 9.0 pka 10.5 pka 9.0 pka 10.5 +N 3 +N 3 +N 3 N 2 +N 3 +N 3 +N 3 N 2 pi 9.8 [-] [-] pi 9.8 [-] [-] Lysine becomes increasingly positive Lysine becomes increasingly negative - 2N - +N 3 +N 3 N 2 4
[-] [-] pka 4.3 [-] [-] +1 +1 0 - [-] pka 4.3 pka 9.7 [-] [-] pka 4.3 pka 9.7 [-] +1 0-1 +1 0-1 -2 - - - - 2N - - - - [-] pka 4.3 pka 9.7 [-] pi 3.3 [-] [-] pi 3.3 +1 0-1 -2 - - 2N - Glutamate becomes increasingly + Glutamate becomes increasingly negative (-) - 2N - - - - - 5
pi 3.3 [-] [-] harges on proteins The formation of the peptide bond neutralises the carboxyl and amino group charges on the alpha carbon BUT not the charges on the side chains. Not all side chains have a charge (only lys, arg, his, glu and asp) Amino acid sequence dependent The charge on Proteins harges on proteins + 3N - R 1 + 3N + + 3N R 2 2 N - - Different proteins have different native charges. The overall charge on a protein will depend on: The sequence The p R 1 R 2 Peptide bond Determining the pi of a protein It can be predicted from the difference between the sum of the acidic side chains (asp + glu) and the sum of the basic side chains (lys + arg + his). It is determined experimentally by techniques such as isoelectric focusing. The protein is placed in a p gradient and subjected to an electric field. The protein moves to its pi. Determining the pi of a protein Those proteins with more acidic residues will have a lower pi Those proteins with more basic residues will have a higher pi. 6
Estimating the charge of a protein Estimating the charge of a protein What we really want to know is the charge of a protein at a particular p, like 7. ow do we use pi data to predict the charge of our protein? Acidic residues lower the pi Basic residues raise the pi. [-] Protein becomes increasingly +ve pi ~5 Protein becomes increasingly -ve [-] Estimating the charge of a protein Estimating the charge of a protein At p 3 the protein will be +ve At p 7 the protein will be -ve [-] p ~3 pi ~5 [-] [-] pi ~5 p ~7 [-] Protein becomes increasingly +ve Protein becomes increasingly -ve Protein becomes increasingly +ve Protein becomes increasingly -ve At a particular p.. If the p of the environment is below (more acidic >) the pi then the protein will be positive (+ve) If the p of the environment is above (more basic >[-]) the pi then the protein will be negative (-ve). Relevance to Molecular Biology If you want a protein to interact with nucleic acid (which has a sugar-phosphate -ve backbone) you will need a protein which has positive charges, at least in the area of the protein where the interaction occurs. Examples are histones, proteins which pack the DNA. These proteins have high pis. 7
Relevance to Molecular Biology Proteins that interact with nucleic acids often use lysine and arginine residues to interact with the sugar-phosphate backbone. Glutamates are often involved with the interactions with the bases. 8