Biochemistry 1.3 Lectures - PDF

Biochemistry 1.3 Dr. Oliver Joyce 2 October - 6 October Lectures by Dr. Oliver Joyce Dr. Oliver Joyce Titration of Alanine At high pH the ionizable groups are unprotonated – anionic form At mid pH range the zwitterionic form dominates At low pH all ioniz...

Biochemistry 1.3 Dr. Oliver Joyce 2 October - 6 October Lectures by Dr. Oliver Joyce Dr. Oliver Joyce Titration of Alanine At high pH the ionizable groups are unprotonated – anionic form At mid pH range the zwitterionic form dominates At low pH all ionizable groups are protonated – cationic form Titration of Alanine From the graph of the titration of alanine, there are 3 values highlighted pK1, pIAla & pK2 pK1 – the pH at which the concentrations of the cationic form & the zwitterionic form of alanine exist in equal measure The pI is the pH at which the molecule carries no nett charge is known as the isoelectric point – Only the zwitterionic form exists pK2 - the pH at which the concentrations of the the zwitterionic form & the anionic form of alanine exist in equal measure  Acid-Base Properties of Amino Acids Key Points: The -COOH and -NH2 groups in amino acids are capable of ionizing (as are the acidic and basic R-groups of the amino acids). At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. As a general rule – the pK1 is at pH~2.0 – the pK2 is at pH~9.4  Acid-Base Properties of Amino Acids The nett charge (the sum of all the charged groups present) of any amino acid, peptide or protein, is dependent on the pH of the surrounding aqueous environment. As the pH of a solution of an amino acid or protein changes so too does the net charge. – This phenomenon can be observed during the titration of any amino acid or protein.  Acid-Base Properties of Amino Acids When the nett charge of an amino acid or protein is zero the pH will be equivalent to the isoelectric point: pI. pI=(pK1+pK2)/2 Acid Base properties of Polypeptides Polypeptides have – free amino group at the N-terminus – free carboxyl group at the C-terminus – These can both ionize & act as weak acids & bases Polypeptides as Polyampholytes In addition polypeptides usually contain some amino acids that have ionizable groups on their side chains. – Lysine – Arginine – Histidine – Glutamic Acid – Aspartic Acid – Cysteine – Tyrosine Polypeptides as Polyampholytes The presence of both positive and negative charges on side chains makes polypeptides polyampholytes Polyampholyte – – A macromolecule that has both anionic & cationic character These various groups have a wide range of pK values, as shown in the next table, but are all weakly acidic or basic groups. Protein Structure varies with pH Changing pH is of critical importance in protein biochemistry. Even a small shift in pH will significantly alter the charges on a protein and may modify its behaviour – N.B. e.g the solubility of a protein is minimal at its isoelectric point. The fact that different proteins have different net charges at a given pH is often used to separate proteins by electrophoresis or ion-exchange chromatography.  Biochemistry How are amino acids joined to form polypeptides? Dr. Oliver Joyce. Lectures by Dr. Oliver Joyce Dr. Oliver Joyce  How are amino acids linked to form proteins? How are amino acids linked to form proteins? Peptide Bonds – Amino acids can be joined together to form a peptide or polypeptide. – They are called peptides because when the carboxyl group of one amino acid joins to the amino group of another, a peptide bond is formed. How are amino acids linked to form proteins? A peptide bond is formed by a condensation reaction This condensation reaction eliminates water As each amino acid is added to the chain, another molecule of water is eliminated The portion of each amino acid remaining in the chain is called an amino acid residue Peptide Bond Formation N.B. Peptide Bond Formation Two amino acids joined = dipeptide. Three amino acids joined = tripeptide. Four to 10 amino acids joined = oligopeptide More than 10 amino acids joined = polypeptide. Peptide Bond Formation Polypeptide refers to the structure of a single chain of amino acids – the term polypeptide is often confused with the term protein – A protein consists of one or more polypeptide chains Peptide Bond Formation Every polypeptide has – one free amino group called the "N-terminal” – one free carboxyl group called the "C-terminal Polypeptide  Amino Acid residues Once an amino acid has been covalently linked to form a polypeptide it is referred to as an amino acid residue (usually abbreviated to residue). Therefore, it is incorrect to say “50 amino acid protein”; the correct usage is “50- residue protein”. The sequence of a polypeptide is usually written using the 3 or 1 letter abbreviation and always with the N-terminus to the left and the C terminus to the right.  Biochemistry Amino Acid residue sequence in polypeptides Dr. Oliver Joyce. Lectures by Dr. Oliver Joyce Dr. Oliver Joyce Amino Acid residue sequence of a Protein To begin to study a protein it is first necessary to prepare the protein in pure form, – free of contamination from any other protein. The next step after purifying a protein is to determine its amino acid residue composition – i.e. the relative amounts of the different amino acids in the protein Composition determinations give limited info about a protein. Much more important is the sequence of the amino acid residues.  Amino Acid residue sequence of a Protein A Polypeptide has direction N terminus to C terminus example: – N-Y-Q-W-C – There is no set length of a polypeptide – however most polypeptides in nature are between 50 and 2000 residues long. – So how do we determine the sequence of a polypeptide? One amino acid at a time! Amino Acid residue sequence of a Protein Frederick Sanger determined the first complete amino acid sequence of a protein in 1953 – The protein was insulin – Insulin is made of 2 polypeptide chains Amino Acid residue sequence of a Protein Insulin Fig 1. Fig 2.  Biochemistry Amino Acid residue sequence Edman Degradation Dr. Oliver Joyce. Lectures by Dr. Oliver Joyce Dr. Oliver Joyce Amino Acid residue sequence of a Protein -Edman Degradation The most important of the sequencing techniques is based on the Edman degradation reaction. Involves a series of reactions that removes residues from a polypeptide chain one at a time starting from the N-terminus. A polypeptide can be read starting from the N- terminal end. Amino Acid residue sequence of a Protein -Edman Degradation To determine the amino acid residue sequence of a protein, – Prior to sequencing peptides it is necessary to eliminate disulfide bonds within peptides and between peptides using 2-mercaptoethanol. – the N-terminal amino acid is labelled for easy detection – the N-terminal amino acid is removed in such a way that the rest of the polypeptide chain remains intact. – The process is continued amino acid by amino acid, – and the amino acid sequence is therefore determined in the N ➔ C direction. Amino Acid residue sequence of a Protein -Edman Degradation  Amino Acid residue sequence of a Protein -Edman Degradation Edman Degradation: sequential removal of amino terminal amino acid using phenyl iso thio cyanate. The process is automated. Amino Acid residue sequence of a Protein -Edman Degradation  Biochemistry Amino Acid residue sequence Proteolytic Enzymes Dr. Oliver Joyce. Lectures by Dr. Oliver Joyce Dr. Oliver Joyce Amino Acid residue sequence of a Protein -Edman Degradation Edman degradation is not reliable for long proteins – so proteins need to be cut into shorter pieces. – This is achieved using proteolytic enzymes – proteolytic enzymes are proteins that ‘cut’ other proteins Amino Acid sequence of a Protein Due to the limitations of the Edman degradation technique, peptides longer than around 50 residues can not be sequenced completely! Amino Acid sequence of a Protein -Edman Degradation Trypsin: cuts carboxyl terminal side of LYS, ARG. But only if the next residue is not Pro Amino Acid sequence of a Protein -Edman Degradation Trypsin: cuts carboxyl terminal side of LYS, ARG. But only if the next residue is not Pro Amino Acid sequence of a Protein -Edman Degradation Chymotrpsin: cuts carboxyl terminal of aromatic amino acids. But only if the next residue is not Pro + Other techniques to keep in mind: Cyanogen bromide (CNBr): – This reagent causes specific cleavage at the C- terminal side of Met residues. – The number of peptide fragments that result from CNBr cleavage is equivalent to one more than the number of Met residues in a protein.