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Charges on amino acids and proteins Charges on amino acids and proteins

Charges on amino acids and proteins - PDF document

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Charges on amino acids and proteins - PPT Presentation

1 Acidic side chainsglutamate and aspartate AHA HCOOHCOOH H Basic side chainsarginine lysine histidine BHB HNHNHH H Glycine pH 1 NCHC H OHThe amino group pKa96The carboxyl ID: 285016

1 Acidic side chains:glutamate and aspartate AHA-

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1 Charges on amino acids and proteins Acidic side chains:glutamate and aspartate AHA- + H+COOHCOO-H+ H + Basic side chains:arginine, lysine & histidine BH+B + H++NHNHH+ H + Glycine@ pH 1 N+CHC H OHThe amino group, pKa9.6The carboxyl group, pKa2.2Overall charge +1 Glycine@ the pKaof the carboxyl group, pH 2.250% in theAcid form, HA 50% in the conjugate Base form, A- N+CHCOHN+CHC H O- Glycine@ pH 7The amino group, +1The carboxyl Group, -1 N+CHC H O-Overall charge, 0 2 Glycine@ the pKaof the amino group, pH 9.650% in the acid form50% in the conjugate base form N+CHCO-NCHC H O- Glycine@ pH 12 NCHC H O-Overall charge, -1 The pI•The pIis the pH where the charge on the amino acid is exactly zero.•If the amino acid is placed in an electric field at this pH it will not move.•If you place the amino acid in a solution at a higher pH it will be negative.•If the solution has a lower pH the amino acid will be positive. Calculating the pIof Glycine•Find the range of pHs where the overall charge of the majority of molecules is zero•In the case of glycinethis is between the pKas2.2 and 9.6.•Find the average = 5.9 p H pH 1[H+] pH 14[OH-][OH-] [H+] pH 7 [H+] = [OH-] Increasing [H+]acidic Increasing [OH-]basic The pI of gly cine [H+] [OH-][OH-] [H+] pKa2.2 +1 N+CHC H OH 3 The pI of gly cine [H+] [OH-][OH-] [H+] pKa9.6 pKa2.2 +1 0 N+CHCOH N+CHCO- pKa 2.2 The pI of gly cine [H+] [OH-][OH-] [H+] pKa9.6 pKa2.2 N+CHCOH N+CHCO- NCHCO- O pKa 2.2 pKa 9.6 +1 0 -1 The pI of gly cine [H+] [OH-][OH-] [H+] pKa9.6 pKa2.2 pI5.9 N+CHCOH N+CHCO- NCHCO- O pKa 2.2 pKa 9.6 +1 0 -1 The pI of gly cine [H+] [OH-][OH-] [H+] pI5.9 Glycinewill be positive (+) Glycinewill be negative (-) N+CHC H OH NCHC H O- The pI of gly cine [H+] [OH-][OH-] [H+] pI5.9 + - The pI of basic amino acids [H+] [OH-][OH-] [H+] pKa2.2 +2 N+CHCCHOHCHCHCH+NH 4 The pI of basic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa9.0 +2 +1 N+CHCCHOHCHCHCH+NH N+CHCCHO-CHCHCH+NH pKa 2.2 The pI of basic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa9.0 pKa10.5 +2 +1 0 N+CHCCHOHCHCHCH+NH N+CHCCHO-CHCHCH+NH NCHCCHO-CHCHCH+NH pKa 2.2 pKa 9.0 The pI of basic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa9.0 pKa10.5 N+CHCCHOHCHCHCH+NH N+CHCCHO-CHCHCH+NH NCHCCHO-CHCHCH+NHNCHCCHO-CHCHCHNH pKa 2.2 pKa 9.0pKa 10.5 +2 +1 0 -1 The pI of basic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa9.0 pKa10.5 N+CHCCHOHCHCHCH+NH N+CHCCHO-CHCHCH+NH NCHCCHO-CHCHCH+NHNCHCCHO-CHCHCHNH pKa 2.2 pKa 9.0pKa 10.5 Overall Charge 0pI9.8 The pI of basic amino acids [H+] [OH-][OH-] [H+] pI9.8 Lysine becomes increasingly positive Lysine becomes increasingly negative NCHCCHO-CHCHCHNH N+CHCCHOHCHCHCH+NH N+CHCCHO-CHCHCH+NH The pI of basic amino acids [H+] [OH-][OH-] [H+] pI9.8 5 The pI of acidic amino acids [H+] [OH-][OH-] [H+] pKa2.2 +1 N+CHCCHOHCHOH The pI of acidic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa4.3 +1 N+CHCCHOHCHOHN+CHCCHO-CHOH The pI of acidic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa9.7 pKa4.3 0 +1 -1 N+CHCCHOHCHOHN+CHCCHO-CHOH N+CHCCHO-CHO- The pI of acidic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa9.7 pKa4.3 0 N+CHCCHOHCHOHN+CHCCHO-CHOH N+CHCCHO-CHO- NCHCCHO-CHO- +1 -1 -2 The pI of acidic amino acids [H+] [OH-][OH-] [H+] pKa2.2 pKa9.7 pKa4.3 0 N+CHCCHOHCHOHN+CHCCHO-CHOH N+CHCCHO-CHO- NCHCCHO-CHO- +1 -1 -2 pI3.3 The pI of acidic amino acids [H+] [OH-][OH-] [H+] pI3.3 Glutamate becomes increasingly negative (-) Glutamate becomes increasingly + N+CHCCHOHCHOH NCHCCHO-CHO- N+CHCCHO-CHO- 6 The pI of acidic amino acids [H+] [OH-][OH-] [H+] pI3.3 Charges 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, gluand asp)•Amino acid sequence dependent The charge on Proteins NCHCO-NCHCO-NCH C R1 ON CHCO- H H2O Peptide bond Charges on proteins•Different proteins have different native charges.•The overall charge on a protein will depend on:–The sequence–The pH Determining the pIof 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 isoelectricfocusing. The protein is placed in a pH gradient and subjected to an electric field. The protein moves to its pI. Determining the pIof a protein•Those proteins with more acidic residues will have a lower pI•Those proteins with more basic residues will have a higher pI. 7 Estimating the charge of a protein•What we really want to know is the charge of a protein at a particular pH, like 7.•How do we use pIdata to predict the charge of our protein?•Acidic residues lower the pI•Basic residues raise the pI. Estimating the charge of a protein [H+] [OH-] [H+] pI~5 Protein becomes increasingly -ve Protein becomes increasingly +ve [OH-] Estimating the charge of a protein [H+] [OH-] [H+] pI~5 Protein becomes increasingly -ve Protein becomes increasingly +ve [OH-] pH ~3At pH 3 the protein will be +ve Estimating the charge of a protein [H+] [OH-] [H+] pI~5 Protein becomes increasingly -ve Protein becomes increasingly +ve [OH-] At pH 7 the protein will be -vepH ~7 At a particular pH..•If the pH of the environment is below (more acidic �[H+]) the pIthen the protein will be positive (+ve)•If the pH of the environment is above (more basic �[OH-]) the pIthen 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 -vebackbone) 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. 8 Relevance to Molecular Biology•Proteins that interact with nucleic acids often use lysine and arginineresidues to interact with the sugar-phosphate backbone.•Glutamates are often involved with the interactions with the bases.