Biochem 1 2014 Lecture #2

Amino Acids, Peptides, Proteins

• Structure and naming of amino acids

• Structure and properties of peptides

• Ionization behavior of amino acids and peptides

• Methods to characterize peptides and proteins

Learning goals:

Overview of amino acid biosynthesis

All amino acids are derived from

intermediates in:

• glycolysis

• the citric acid cyle

• pentose phosphate pathway

Nitrogen enters these pathways

by way of glutamate and

glutamine.

Proteins: Main Agents of Biological Function

•Receptors

-Signal transduction

- receptor mediated endocytosis

•Catalysis

– enolase (in the glycolytic pathway)

– DNA polymerase (in DNA replication)

• Transport

– hemoglobin (transports O2 in the blood)

– lactose permease (transports lactose across the cell membrane)

• Structure

– collagen (connective tissue)

– keratin (hair, nails, feathers, horns)

• Motion

– myosin (muscle tissue)

– actin (muscle tissue, cell motility)

Amino Acids: Building Blocks of Protein

• Proteins are linear heteropolymers of -amino acids

• Amino acids have properties that are well-suited to

carry out a variety of biological functions

– Capacity to polymerize

– Useful acid-base properties

– Varied physical properties

– Varied chemical functionality

Amino acids share

many features,

differing only at the

R substituent

General structure of an amino

acid. This structure is common to

all but one of the α-amino acids.

(Proline, a cyclic amino acid, is the

exception.) The R group, or side-

chain (red), attached to the α

carbon (blue) is different in each

amino acid.

Most -amino acids are chiral

• The -carbon always has four substituents

and is tetrahedral

• All (except proline) have:

– an acidic carboxyl group

– a basic amino group

– an -hydrogen connected to the -carbon

• The fourth substituent (R) is unique

– In glycine, the fourth substituent is also hydrogen

Amino Acids: Atom Naming

• Organic nomenclature: start from one end

• Biochemical designation:

– start from -carbon and go down the R-group

All amino acids are chiral (except glycine)

Proteins only contain L amino acids Stereoisomerism in α-amino acids. (a) The two

stereoisomers of alanine, L- and D-alanine, are

nonsuperposable mirror images of each other

(enantiomers). (b, c) Two different conventions

for showing the configurations in space of

stereoisomers. In perspective formulas (b) the

solid wedge-shaped bonds project out of the

plane of the paper, the dashed bonds behind it. In

projection formulas (c) the horizontal bonds are

assumed to project out of the plane of the paper,

the vertical bonds behind. However, projection

formulas are often used casually and are not

always intended to portray a specific

stereochemical configuration.

Amino Acids: Classification

Common amino acids can be placed in five

basic groups depending on their R substituents:

• Nonpolar, aliphatic (7)

• Aromatic (3)

• Polar, uncharged (5)

• Positively charged Basic (3)

• Negatively charged Acidic (2)

The 20 common amino acids of

proteins The structural formulas show the

state of ionization that would

predominate at pH 7.0. The

unshaded portions are those

common to all the amino acids;

the portions shaded in pink are

the R groups. Although the R

group of histidine is shown

uncharged, its pKa, is such that a

small but significant fraction of

these groups are positively

charged at pH 7.0

These amino acid side chains absorb UV light at 270–280 nm

These amino acids side chains can form hydrogen bonds.

Cysteine can form disulfide bonds.

Uncommon Amino Acids in Proteins

• Not incorporated by ribosomes

except for Selenocysteine

• Arise by post-translational modifications of

proteins

• Reversible modifications, especially

phosphorylation, are important in regulation

and signaling

Modified Amino Acids Found in Proteins

Reversible Modifications of Amino Acids: these modifications are for the

purpose of protein regulation, the most common is PO4

Ionization of Amino Acids

• At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form.

• At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions.

• At alkaline pH, the amino group is neutral –NH2 and the amino acid is in the anionic form.

Cation Zwitterion Anion Titration of an amino acid.

Shown here is the titration

curve of 0.1 M glycine at

25°C. The ionic species

predominating at key points

in the titration are shown

above the graph. The

shaded boxes, centered at

about pK1 = 2.34 and pK2 =

9.60, indicate the regions of

greatest buffering power.

Note that 1 equivalent of OH–

= 0.1 M NaOH added.

Chemical Environment Affects pKa Values

-carboxy group is much more acidic than in carboxylic acids -amino group is slightly less basic than in amines

The pKa values for the ionizable groups in glycine are lower than those for

simple, methyl-substituted amino and carboxyl groups. These downward

perturbations of pKa are due to intramolecular interactions. Similar effects can be

caused by chemical groups that happen to be positioned nearby—for example,

in the active site of an enzyme.

Amino acids can act as buffers

Amino acids with uncharged side chains, such as

glycine, have two pKa values:

The pKa of the -carboxyl group is 2.34

The pKa of the -amino group is 9.6

Glycine can act as a buffer in two pH regimes.

Buffer Regions

Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at

25°C. The ionic species predominating at key points in the titration = 2.34 and

pK2 = 9.60, indicate the regions of greatest buffering power. Note that 1 equivalent

of OH– = 0.1 M NaOH added. are shown above the graph. The shaded boxes,

centered at about pK1= 2.34 and pK2 = 9.60, indicate the regions of greatest

buffering power. Note that 1 equivalent of OH– = 0.1 M NaOH added.

Amino acids carry a net charge of zero at a specific pH (the pI)

• Zwitterions predominate at pH values between the pKa

values of the amino and carboxyl groups

• For amino acids without ionizable side chains, the Isoelectric

Point (equivalence point, pI) is:

• At this point, the net charge is zero

– AA is least soluble in water

– AA does not migrate in electric field

2

21 pKpKpI

Ionizable side chains can show up in titration curves

• Ionizable side chains can be also titrated

• Titration curves are more complex

• pKa values are discernable if two pKa values are

more than two pH units apart

Why is the side chain pKa so much higher?

How to Calculate the pI When the Side Chain is Ionizable

• Identify species that carries a net zero charge

• Identify pKa value that defines the acid strength of

this zwitterion: (pK2)

• Identify pKa value that defines the base strength of

this zwitterion: (pK1)

• Take the average of these two pKa values. What

is the pI of histidine?

Formation of Peptides • Peptides are small condensation products of amino acids

• They are “small” compared to proteins (Mw < 10 kDa)

• The α-amino group of one amino acid (with R2 group) acts as a nucleophile to displace the hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced. At physiological pH, the reaction shown here does not occur to any appreciable extent.

Peptide ends are not the same

Numbering (and naming) starts from the amino terminus

AA1 AA2 AA3 AA4 AA5

Naming peptides: start at the N-terminus

• Using full amino acid names

– Serylglycyltyrosylalanylleucine

• Using the three-letter code abbreviation

– Ser-Gly-Tyr-Ala-Leu

• For longer peptides (like proteins) the one- letter code can be used

– SGYAL

Polypeptide size and number varies greatly in proteins

Classes of Conjugated Proteins

What to Study about Peptides and Proteins

What is its sequence and composition?

What is its three-dimensional structure?

How does it find its native fold?

How does it achieve its biochemical role?

How is its function regulated?

How does it interacts with other macromolecules?

How is it related to other proteins?

Where is it localized within the cell?

What are its physico-chemical properties?

A mixture of proteins can be separated

• Separation relies on differences in physical and chemical properties

– Charge

– Size

– Affinity for a ligand

– Solubility

– Hydrophobicity

– Thermal stability

• Chromatography is commonly used for preparative separation

Column Chromatography

Separation by Charge

Separation by Size

Separation by Affinity

Electrophoresis for Protein Analysis

Separation in analytical scale is commonly done by electrophoresis

– Electric field pulls proteins according to their charge

– Gel matrix hinders mobility of proteins according to their size and shape

SDS PAGE: Molecular Weight

• SDS – sodium dodecyl sulfate – a detergent

• SDS micelles bind to and unfold all the proteins

– SDS gives all proteins an uniformly negative charge

– The native shape of proteins does not matter

– Rate of movement will only depend on size: small proteins will move faster

SDS-PAGE can be used to calculate the molecular weight of a protein

Isoelectric focusing can be used to determine the pI of a protein

Isoelectric focusing and SDS-PAGE are combined in 2D electrophoresis

Spectroscopic Detection of Aromatic Amino Acids

• The aromatic amino acids absorb light in the UV region

• Proteins typically have UV absorbance maxima around

275–280 nm

• Tryptophan and tyrosine are the strongest

chromophores

• Concentration can be determined by UV-visible

spectrophotometry using Beers law: A = ·c·l

Specific activity (activity/total protein) can be used to assess protein purity

Protein Sequencing

• It is essential to further biochemical analysis that we know the sequence of the protein we are studying

• Actual sequence generally determined from DNA sequence

• Edman Degradation (Classical method)

– Successive rounds of N-terminal modification, cleavage, and identification

– Can be used to identify protein with known sequence

• Mass Spectrometry (Modern method)

– MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence

– Can be used to determine post-translational modifications

Edman’s Degradation

MS Procedures for Sequence IDs

Protein Sequences as Clues to Evolutionary Relationships

• Sequences of homologous proteins from a wide range of species can be aligned and analyzed for differences

• Differences indicate evolutionary divergences

• Analysis of multiple protein families can indicate evolutionary relationships between organisms, ultimately the history of life on Earth