BIOL 1510 Chapter 3

Standard structure of an amino acid

chiral carbon, carboxylate group, amino group, R group

What enantiomer of amino acids does the body use?

L form

What definition of acids and bases are we using in biochem?

Bronsted-Lowry

How many pKa points do amino acids have?

Usually two but can have three if the R group is ionizable

How do electron withdrawing group affect acidity?

Increase acidity and lower pKa

Describe the progression of a titration of a normal amino acid

1. At low pH, only the amino group is protonated

2. As you add base and reach the pI point, both the amino group and carboxylate group are charged

3. As you keep adding base, only the carboxylate group is deprotonated

Isoelectric point

Where amino acid exists as its zwitterion-neutral molecule!

Describe the progression of a titration of an acidic amino acid

1. At low pH, only the backbound amino group is protonated

2. As you add base and reach the pI point, both the backbone amino group and backbone carboxylate group are charged

3. As you keep adding base the acidic R group gets deprotonated in addition to the backbone groups that are already charged

4. As you add your final equivalents of base, both the backbond COOH and R group COOH are deprotonated and the backbone amino group is not charged

What kind of reaction energetically is peptide bond formation?

Anabolic and endergonic

What kind of reaction in terms of water going in/out of the bond is peptide bond formation?

dehydration otherwise known as condensation reaction

Stereochemistry of peptide bond

planar

Disulfide bond

A bond formed between two cysteins

Protein properties one can take advantage of to separate proteins

• size

• shape

• charge

• hydrophobicity/hydrophilicity

• binding affinity

• protein modifications

Technique(s) for separating proteins

Chromatography

Chromatography

Separation of proteins using a mobile and stationary phase in a column

Types of chromatography

• ion exchange

• gel filtration/exclusion

• affinity

Ion exchange chromatography

Stationary phase resin is coated with a charge and oppositely charged proteins cling to the stationary phase

Gel filtration/exclusion chromatography

Stationary phase resin has pores so smaller proteins get stuck while larger proteins can wash through

Affinity chromatography

Stationary phase resin is coated with a ligand or antibody that is specific to the protein of interest

Ways to check effectiveness of purification

Look at activity of purified protein(s)

• relate yield to overall activity

• relate purity to specific activity

Technique(s) to characterize proteins

• electrophoresis/SDS-PAGE

• isoelectric focusing

• 2D PAGE

SDS-PAGE

A way to determine proteins’ molecular weight

• SDS is a detergent that denatures proteins and coats them with an even negative charge

Isoelectric focusing

A way to determine proteins’ charge

• set up an electric and pH gradient

• protein will stop at the pH of its isoelectric point

2D PAGE

A combination of SDS-PAGE and isoelectric focusing that helps determine proteins’ size and charge

1. do isoelectric focusing to determine charge

2. do SDS-PAGE to determine size

Primary protein structure

amino acid sequence

Secondary protein structure

Basic 3D chape cause by interactions between nearby resides

• alpha helices

• beta sheets

Tertiary protein structure

complex 3D structure due to covalent and non-covalent interactions between far away resides

Quaternary structure

Multiple peptides come together

What determines protein structure and therefore function?

amino acid sequence

Composition

The type and number of each amino acid present in a protein

Sequence

The order of amino acids in a protein

How do you determine amino acid composition?

Boil protein down into its parts in an acidic solution and do chromatography

How do you determine amino acid sequence?

Edman degradation OR sequence the gene itself

How can you compare related proteins between species in terms of amino acid composition?

A box plot that shows the relative percent of sequences that have a particular residue at a particular spot in the protein’s sequence

How can you look at evolutionary divergence using proteins?

Look at the alignment of related proteins between species

Aromatic amino acids

Phenylalanine, Tyrosine, Tryptophan