IV Proteins A. Amino acids (a.a.) 1. Proteins are composed of amino acids covalently bonded to each other in a linear form a- we will see later that this is what is known as the primary sequence of a protein b- an amino acid can be referred to as a “residue” reflecting the loss of a water molecule that occurs when two amino acids bond to each other 2. Each a.a. is centered around a central carbon atom designated the α-carbon a- this differs from the typical numbering of carbons b- other carbons in the a.a. are then named using the Greek alphabet

3. The α-carbon is a chiral center a- in all amino acids except for glycine the α-carbon is bonded to four different groups 1) a carboxyl group 2) an amino group 3) a hydrogen atom 4) a variable R group b- in glycine the R group is a hydrogen atom c- amino acids have two possible stereoisomers that are nonsuperimposable mirror images of each other known as enantiomers d- the absolute configuration of an amino acid is based on the D,L system which is centered on the two stereoisomers of glyceraldehyde

4. Amino acids found in proteins are mostly in the L configuration a- D-amino acids are rarely in proteins but can be found in other areas of nature - D-amino acids are found in the cell walls of bacteria b- cells almost exclusively produce amino acids in the L configuration for proteins because this configuration allows for the optimum secondary interactions that are necessary for a polypeptide to reach its functioning 3-D configuration - enzyme active site are asymmetrically geared towards this type of production

5. Amino acids can be classified by their R group a- there are 20 amino acids that occur in nature. Each of these 20 are split into 1 of 5 groups: Non polar aliphatic, Aromatic, Negatively charged (Acidic) and Positively charged (Basic) b- the specificity for each protein is determined by its unique sequence of amino acids and the properties of these a.a.’s - ultimately the R group of each amino acid and their unique combinations is where the specificity is coming from because this is the only portion that is different among amino acids 6. Nonpolar aliphatic R group a- Glycine, Alanine, Proline, Valine, Leucine, Isoleucine and Methionine

b- In proteins, nonpolar amino acids tend to cluster together via hydrophobic interaction - mainly alanine, valine, leucine and isoleucine c- since glycine has only a hydrogen atom in its R group it does not play a large role in hydrophobic interactions d- due to proline’s cyclic imino (secondary amino) residue, it adds a lot of rigidity to regions of proteins that contain proline

7. Aromatic a- phenylalanine, tyrosine and tryptophan b- all are mainly nonpolar but tyrosine and tryptophan can form some hydrogen bonds. This makes these two a.a.’s more polar than phenylalanine. - Looking at there respective R groups can you see why this would be the case?

c- aromatic amino acids absorb UV light which allows protein levels to be monitored at a wavelength of 280nm. - spectrophotometers can thus be used to measure protein levels

8. Polar uncharged R group a- serine, threonine, cysteine, asparagine and glutamine b- these amino acids are soluble in water thus are referred to as hydrophilic c- they are capable of forming H-bonds with water d- serine and threonine have hydroxyl groups in their R group cysteine has a sulfhydryl group asparagine and glutamine have amide groups e- cysteine is capable of forming a very hydrophobic covalent disulfide bond with itself that can add a lot of stability to a protein structure

9. The positively charged amino acids (Basic) a- lysine, arginine, and histidine b- very hydrophilic c- have a positive charge at pH 7 d- histidine residues are frequently involved in enzyme catalysis because its R group is ionizable near neutrality (pKa= 6.0)

10. the negatively charged amino acids a- aspartate and glutamate b- both have a net negative charge at pH 7

11. There are other amino acids that can be found in proteins in in rare cases. a- these amino acids are often derivatives of 1 of the 20 common a.a.’s b- 4-hydroxyproline is a derivative of proline that can be found in plant cell walls and in collagen c- 5-hydroxylysine is also found in collagen 6-N-methyllysine is a key component of myosin γ-carboxyglutamate is important in the blood clotting protein prothrombin and calcium ion binding proteins desmosine is found in the fibrous protein elastin Selenocysteine a rare amino acid that is made during protein synthesis rather than through postsynthetic modification

B. Amino acids as acids and bases 1. When amino acids are dissolved in water they can exist in solution as a dipolar ion known as a zwitterion a- the amino group and the carboxyl group of every amino acid can donate a proton b- alanine can be described as a monoamino monocarboxylic α-amino acid that is a diprotic acid when fully protonated c- thus zwitterions can act as acids (proton donors) or as bases (proton acceptors) depending on the pH of the solution and their amount of protonation

Zwitterion as a proton donor- H H R C NH 3 + COO - R C NH 2 COO - + H + H R C NH 3 + COO - + H + Zwitterion as a proton acceptor- R C NH 3 + COOH H

R C NH 3 + COO - H R C NH 2 COO - H R C NH 3 + COOH H H+H+ H+H+ Net Charge:

2. Titration curves of amino acids a- because amino acids are (at least) diprotic their titration curves appear a little different from those we have seen to this point -each proton will have a pKa value and thus there are two stages in the titration curve b- depending on where in the titration you are looking (i.e. which pH) a different form of the amino acid will be prevalent c- remember that pH is notation for proton concentration and that pKa is the equilibrium constant for ionization - thus pKa is a measure of the tendency for a group to give up a proton - as the pKa increases by one unit the tendency to give up the proton decreases tenfold

d- the inflection point pI is the point when removal of the first proton is complete and he second has just begun so the amino acid’s prevalent form is as a dipolar ion e- the titration of glycine shows that it has two buffering regions centered around its two pKa values - within these regions the Henderson-Hasselbalch equation can be used to identify the proportion of proton-donor and proton acceptor species needed of glycine for a given pH

f- the pKa value for a given functional group ( i.e. its tendency to give up electrons) is greatly affected by its chemical environment - a comparison of the pKa for the carboxyl portion of glycine (pKa= 2.43) versus the carboxyl of acetic acid (pKa= 4.76) shows this point. In glycine the positively charged amino group attached to the α-carbon helps to push the departing proton of the carboxyl group out more easily - at the α-amino group the electronegative atoms of oxygens on the carboxyl help to pull the hydrogen ion (proton) away (pKa= 9.6). This is not true for methylamine (pKa= 10.6). - these differences have a major impact in biology in terms of facilitating chemical reactions at an enzymes active site by exploiting different pKa values of various amino acid residues acting as proton donors or acceptors

g- the isoelectric point or isoelectric pH or pI - this is the point when the net charge of an amino acid is zero; the point where the dipolar form is predominant - for a.a.’s that do not have an ionizable R group the pI = ½ (pK1 + pK2) - any pH below the pI and the net charge of the amino acid will be positive and pH above and it will be negative - positive charge migrates towards the anode (positive electrode), negative charge migrates towards the cathode (negative electrode); the greater the distance from the pI the greater the net charge - using this information the net charge of an amino acid can be predicted for any pH

h- when an amino acid has an ionizable R group there will be three pKa values. - to calculate the pI you must identify the two values that saddle the point on the titration curve when the net charge of the amino acid is zero - the differences in pI values for different amino acids are an indication of the different acidic and basic properties they have j- the pKa’s for the carboxyl group of most amino acids falls in the range of 1.8 – 2.4 while the pKa for the amino groups tend to fall in the range of 8.8 – cysteine, histidine, aspartate and glutamates R group pKa’s fall between their carboxyls’ and aminos’ - Tyrosine, Lysine and Arginine R group pKa’s are above their amino pKa