CH 5 The Primary Level of Protein Structure

HW 2, 3, 4, 6, 7

Amino Acids and Peptides

• Bioimportance:– Monomer units for proteins– Participate in cellular functions such as nerve

transmissions– Biosynthesis of porphyrins, purines,

pyrimidines, and urea

• While human proteins only contain L-amino acids, other organisms contain both D and L

• 10 of the 20 amino acids commonly used are essential nutrients, which mean they must be in our diet, we can not synthesize them

• There are over 300 naturally occurring amino acids, but only 20 are used to make proteins.

• These 20 are listed in table 5.3 page 129, according to side chain properties

• Friday, we will have a quiz on the names, 1 letter abbreviation, and 3 letter abbreviation

• Next Monday, we will have a quiz on the name, structure, and pKa’s (Table 5.1)

• While we only use 20, some of these can be altered in a peptide by adding or removing a functional group to increase diversity.

Chirality

• All amino acids, except Gycine, have an alpha carbon that is chiral.

• This is the source of all chirality in organisms

• The absolute configuration of all the alpha carbons are L

Amino Acids properties

• Amino Acids may have a positive, negative or zero net charge.

• Most amino acids are in the zwitterionic state at physiologic pH

• An amino acid can not exist as COOH/NH2 because any pH low enough to protonate the COO- group would as protonate the NH2

• Some amino acids, histidine and arginine, are resonance hybrids, but they still only have a 1+ charge.

• By altering the net charge via pH, we can create separation processes for amino acids, peptides, and proteins

• The isoelectric species is the form of a molecule that has an equal number of positive and negative charges, thus it is neutral.

• The isoelectric pH, also called pI, is the pH midway between the pKa values on either side of the isoelectric species

• Examples:

• This pI guides selection of separation conditions

• The pKa of side chains varies slightly– Nonpolar effects-

• Thus the pKa in peptides and proteins will depend on unique local environments

• These changes in charge affect physical properties of amino acids, peptides, and proteins.

• Functional groups of the side chains typically determine chemical properties

• When amino acids are in a protein or peptide chain, they are called residues

• Peptides are usually written with the free alpha amino group to the left and the free alpha carboxyl group to the right

• The backbone will start with N, then the alpha carbon, then the carbonyl carbon, then repeat.

• The side chains are bonded to the alpha carbon

• Lines are used with 3-letter abbreviations, omitted with 1-letter abbreviations

• For some peptides, non-common amino acids may be used or non-peptide bonds my be used

• The peptide bond is not charged, however you still have a + at the N-terminal, - at the C-terminal, and any charges on side chains

• Peptides are therefore classified as Polyelectrolytes

Structure Feature of Peptide Bond

• See figure 5.12 page 138• The peptide bond has some double bond

character and does not have rotation about the C-N peptide bond

• The carbonyl O and C, the N,and the H on the N, all lie in the same plane.

• Rotation only occurs between the alpha carbon and N, and alpha carbon and COO-

Determination of Primary Structure• We know proteins are very important

• An important goal of molecular medicine is the identification of proteins who presence, absence, or deficiency is associated with specific physiologic states or disease

• The primary structure of proteins, which is the sequence of amino acids, provides both a molecular finger print for its identification and information that can be used to identify and clone the gene or genes that encode it.

• In order to determine the amino acid sequence, a protein or peptide must be highly purified

• Because of the 1000’s of different proteins in each cell, this is very difficult to do.

• It usually requires many successive purification techniques

Purification

• The classic approach exploits differences in:– Relative solubility of proteins as a function of

pH– Polarity – Salt concentration– Chromatographic separtions

Chromatographic Separations

• Mobile phase vs stationary phase– Paper chromatography– TLC– Column

• Types of stationary phase– Size exclusion– Absorption– Ion exchange

Other types of Chromatography

• pH based chromatography

• Hydrophobic Interaction chromatography

Affinity Chromatography

• Exploits high selectivity of binding proteins

• This is what we did in lab

• All the chromatography mentioned so far, is typically done slowly with low pressure

• The stationary phases involved are somewhat “spongy” and their compressibility limit flow rates

HPLC

• High Pressure Liquid Chromatography uses incompressible silica or alumina as stationary phase which allows much higher flow rates and pressures

• This also helps limit diffusion thus enhances the resolution

• This method is very effective on complex mixtures of lipids or peptides with very similar properties

HPLC

• The stationary phase is typically hydrophobic and water miscible organic solvents such as acetonitrile or methanol are used as mobile phases

SDS-PAGE• SDS- sodium dodecyl sulfate (anion

detergent)

• PAGE- poly acrylamide gel electrophoresis

• Electrophoresis separates biomolecules based on their ability to move through a gel matrix due to an applied electric field

• SDS denatures and binds to proteins at a known ratio of 1 SDS for every 2 peptide bonds

• 2-mercaptoethanol or dithiothreitol is used to break disulfide linkages

• The charge on SDS,-1, overcomes and negates charges on side chains

• This leads to a constant charge to mass ratio which means the peptides are separated purely by the resistance the matrix provides

• Larger peptides have more resistance, therefore move slower

• The gel is then stained, usually with Coomassie Blue, to visualize the movement

IEF• IEF- Isoelectric Focusing

• Ionic buffers called ampholytes and applied electric field are used to generate a pH gradient with in a matrix

• The peptide/protein then migrate through the matrix to an area where the pH=pI, so there is no net charge on the peptide

• IEF can be used in conjunction with SDS-PAGE to perform a 2-D analysis, separating peptides first by pI, then by size

Sequencing Peptides

• Sanger was the first to determine the sequence of a polypeptide

• Mature insulin consist of 2 chains, the A chain has 21 residues, the B chain has 30 residues

• The chains are held together by disulfide linkages

• Sanger first broke the linkages to separate the chains

• He then broke the chains into smaller pieces using trypsin, chymotrypsin, and pepsin

• These reagents cleave peptide bonds at known locations (see table 5.4 p 139)

• These smaller fragments where the separated and hydrolyzed to form even smaller peptide chains

• Each was reacted with 1-fluoro-2,4-dinitrobenzene, called Sanger’s reagent, which derivatizes the exposed alpha amino group

• The amino acid content of the peptide was then determined.

• Working backwards, he was able to determine the complete sequence of insulin and win the Nobel Prize in 1958

Peptide Cleaving Agents

Reagent Bond Cleaved

CNBr Met-X

Trypsin Lys-X and Arg-X

Chymotrypsin Hydrophobic AA-X

Endoproteinase Lys-C Lys-X

Endoproteinase Arg-C Arg-X

Endoproteinase Asp-N X-Asp

Reagent Bond Cleaved

V8 protease Glu-X particularly where

X is hydrophobic

Hydroxylamine Asn-Gly

o-Iodosobenzene Trp-X

Mild Acid Asp-Pro

Edman’s Reagent• Pehr Edman introduced phenylisothiocyanate,

called Edman’s reagent, to selectivity label the amino-terminal residue of a peptide

• Unlike Sanger’s, Edman’s derivative can be removed under mild conditions with out disrupting the rest of the peptide

• After removal, a new amino terminal is produced and the process is repeated

• This allows for the direct sequencing of a peptide

• However, due to the efficiency of the reaction, this process is limited to peptides no larger than 20-30 residues

• Process:

• Because of the limitations to Edman’s process and since most polypeptides contain several hundred residues, most polypeptides must be broken into smaller chains which are then identified.

• Sample problem on hand out.

Microbiology Impact

• Advances in Microbiology have led to a new, simpler way to identify the primary structure of proteins.

• Knowledge of the DNA sequence permits deductions of the sequence of AA

• Sequencing DNA requires much less sample

Example

• So by sequencing only a small portion of protein, we can correlate it to DNA, find the section of DNA that encodes the protein, then deduce the whole sequence.

• Limitation: No information is provided for post-translational modifications!