1

Amino Acid Stereochemistry Chirality and optical activity

Representation of chiral molecules

Fisher Projection

Cahn-Ingold-Prelog

Amino acids and chirality

Modifications and derivatives of amino acids

Protein Structure 4 levels of protein structure

Sequence and primary structure of proteins

Basics of chemical sequencing for proteins

Chapter 4: Stereochemistry Molecular models would be useful for this section!

2

Optical activity - The ability

to rotate plane - polarized light

Asymmetric carbon atom (i.e.,

carbon with 4 different groups)

Chirality - Not superimposable

Mirror image - enantiomers

(+) dextrorotatory - right -

clockwise

(-) levorotatory - left -

counterclockwise

}

Chapter 4: Stereochemistry

3

The Fischer Projection

Representation of the absolute configuration about an

asymmetric carbon.

related to glyceraldehyde

(+) = D-Glyceraldehyde

(-) = L-Glyceraldehyde

4

L-amino acids have NH3+

to the left in a Fischer

projection

There is no direct

relationship between +/-

and D/L or R/S

Racemic a mixture of

equal amounts of a chiral

compound (i.e., 50% R and

50% S)

2N possible enantiomers

Amino Acids with 2 Chiral Centers

5

Cahn - Ingold - Prelog system Can give absolute configuration nomenclature to multiple

chiral centers.

Priority

Atoms of higher atomic number bonded to a chiral center

are ranked above those of lower atomic number.

With the lowest priority away from you R (rectus right/straight) highest to lowest =

clockwise

S (sinistrous/sinistral left) highest to lowest = counterclockwise

SH>OH>NH2>COOH>CHO>CH2OH>C6H5>CH3>H

6

2S,3R 2R,3S

1

2

3

2S,3S 2R,3R

7

Steps for building L-a-amino acids

1. Build NH2-Ca-COOH backbone

2. Place one Ca hydrogen facing away from you.

3. Add sidechain on the hydrogen facing toward you.

4. Examine directionality of N COOH Sidechain

5. Counterclockwise = L-a-amino acid (C-CORN Rule)

6. Clockwise = D-a-amino acid

Counterclockwise

CO R N

= C-CORN Rule

(R)

8

PTMs of Amino Acids Post-translationally

modified amino acids

These transformations are made after the amino acids are already incorporated into a protein

Typical alterations include: hydroxylation, methylation, acetylation, carboxylation, and phosphorylation

Addition of PO32- to a

Ser, Thr, or Tyr is a common theme in signal transduction

9

Molecules Derived from Amino Acids

Neurotransmitters

GABA: glutamate decarboxylation product

Dopamine: tyrosine derivative

Local mediator of allergic reactions

Histamine: histidine decarboxylation product

Thyroid hormone that stimulates vertebrate metabolism

Thyroxine: tyrosine derivative

About 250 amino acids have been found in various plants and fungi

10

Chapter 5 Proteins: Primary Structure

Polypeptide diversity

Protein Purification (later)

Protein sequencing

Preliminary steps

Polypeptide cleavage

Edman degradation

Reconstituting the proteins sequence

Protein evolution

11

4 Levels of Protein Structure

1. Primary structure

1 = Amino acid sequence of the peptide chain(s), the

linear order of AAs.

Remember from the N-terminus to the C-terminus

2. Secondary structure

2 = Local spatial alignment of amino acids without regard

to side chains.

Usually repeated structures

Examples: a-helix, b-sheets, random coil, and b-turns

12

3. Tertiary Structure

3 = the 3-dimensional structure of an entire peptide

(e.g., folding of secondary structural elements against

one another).

Can reveal the detailed chemical mechanisms of an

enzyme.

4. Quaternary Structure

4 = two or more peptide chains associated with a

protein.

Relative spatial arrangements of subunits (separate

polypeptide chains).

13

4 Levels of Protein Structure

14

15

In Addition to Amino Acids In addition to the 20 standard amino acids, some

proteins have something more, such as:

Metal ions (Zn2+, Mg2+, Ca2+, etc.)

Co-enzymes, such as pyridoxal phosphate (vitamin b6

derivative), flavins, NADH, NADPH, etc.

Post-translational modifications, such as,

phosphorylation, addition of carbohydrates,

carbamylation, etc.

Lots of water

These cofactors and coenzymes play both

structural and functional (i.e., catalytic) roles

16

Importance of Protein Sequences

1. Structural studies require sequence information.

2. Sequence comparisons can suggest function

and evolutionary relationships between

proteins.

3. Diseases are often caused by a small number

(1-2, sometimes more) changes (mutations,

mistakes) in a protein sequence. Sequencing of

the DNA (from which protein sequence arises)

of relevant genes can help in the development

of genetically-based drugs.

17

Primary structure of bovine (i.e., cow) insulin. Note the disulfide

bonds connecting the two chains of the protein.

Protein Primary Structure (Sequence)

18

Fred Sanger won the Nobel prize for protein

sequencing of Insulin.

It took 10 years, many people, 100 g of protein!

Current sequencing of the same protein requires a

few micrograms of protein and a few days.

Protein Sequencing: Chemical Sequencing

19

Chemical Protein Sequencing

1. Separate polypeptide chains:

Reduce disulfides, separate individual polypeptide

chains, immobilize on PVDF membrane.

2. Sequencing the peptide chains:

Fragment subunits into smaller peptides 50

AAs in length and purify the fragments.

Determine the sequence of each fragment.

Repeat with different fragmentation system.

3. Assemble sequences:

Organize overlapping sequences, ID modified AAs.

At best the automated instruments can sequence about 50

amino acids in one run!

20

Define by finding end groups.

Bovine insulin should give 2 N-termini and 2 C-termini

N-terminal identifying agent

1-Dimethylaminonaphthalene-5-sulfonyl chloride (Dansyl

chloride)

Reacts with amines: N-terminus + Lys (K) side chains

Detected by its intense yellow fluorescence

How Many Peptides in a Protein?

21

22

CHNH C NH CH NH CH

O

O

Rn-2 Rn-1 Rn

C

O

C

O

CHNH C NH CH

ORn-2 Rn-1

C

O

H3N CH O

Rn

C

O

O

H2O Carboxypeptidase

Carboxypeptidase Cleavage at the C-terminus

Carboxypeptidase A Rn R, K, P Rn-1 P

Aminopeptidases cleave residues from the N-terminus

Carboxypeptidases cleave residues from the C-terminus

Exopeptidases versus Endopeptidases

Carboxypeptidase B Rn= R, K Rn-1 P

23

Cleavage of Disulfide Bonds Permits separation of polypeptide chains and Prevents refolding back to

native structure

Performic acid oxidation

Cystine (-S-S-) or cysteine (-SH) to Cysteic acid (-SO3-)

Methionine to Methionine sulfone, Trp destroyed

2-Mercaptoethanol, dithiothreitol, or dithioerythritol

Keeps the equilibrium toward the reduced form

24

The amino acid composition of a peptide chain is determined by

its complete hydrolysis followed by the quantitative analysis of

the liberated amino acids. Suggestive of protein structure.

Acid hydrolysis (6 N HCl +

Phenol) at 120 oC for 10 to

100 h

destroys Trp and partially

destroys Ser, Thr, and Tyr.

Also Gln and Asn yield Glu

and Asp

Base hydrolysis 2 to 4 N

NaOH at 100 oC for 4 - 8 h.

Is problematic, destroys Cys

Ser, Thr, Arg but does not

harm Trp.

Amino Acid Composition

25

Amino Acid Compositions Suggest

Protein Structures

Leu, Ala, Gly, Ser, Val, Glu and Ile are the most

common amino acids

His, Met, Cys and Trp are the least common.

Polar to non-polar ratios are indicative of globular

or membrane proteins.

Certain structural proteins are made of repeating

peptide structures, i.e., collagen.

26

Amino Acid Analyzer

In order to quantitate the amino acid residues after hydrolysis, each

must be derivatized at about 100% efficiency to a compound that is

colored. Pre- or post-column derivatization can be done.

Separated using HPLC in an automated setup -

each AA has known elution time

CH

CH

O

O

SH CH

2

CH

2

OH

NH3+ C

HCOO-

R

+

+

N

S

CH

COO-

R

CH

2

CH

2

OH

phthalaldehyde

b-mercapto ethanol

amino acid

27

Proteases and Protein Fragmentation

NH CH C

ORn-1

NH CH C

ORn

Scissile Bond

28

Edman Degradation: Phenyl Isothiocyanate, PITC

Edman degradation

used to automatically

sequence the

29

Edman degradation has been automated as a

method to sequence proteins. The PTH-amino acid

is soluble in solvents that the protein is not. This fact

is used to separate the tagged amino acid from the

remaining protein, allowing the cycle of labeling,

degradation, and separation to continue.

Even with the best chemistry, the reaction is about

98% efficient. After sufficient cycles more than one

amino acid is identified, making the sequence

determination error-prone at longer reads.

30

Specific Chemical Cleavage Reagents Cleave the large protein using i.e., trypsin (Rn-1=Lys, Arg), separate

fragments and sequence all of them. (We do not know the order of

the fragments!!)

Cleave with a different reagent i.e., Cyanogen Bromide (Rn-1=M),

separate the fragments and sequence all of them. Align the

fragments with overlapping sequence to get the overall sequence.

31

How to assemble a protein sequence

1. Write a blank line for each amino acid in the

sequence starting with the N-terminus.

2. Follow logically each clue and fill in the blanks.

3. Identify overlapping fragments and place in

sequence blanks accordingly.

4. Make sure that all your amino acids fit into the

logical design of the experiment.

5. Double check your work.

32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 H3N-_-_-_-_-_-_-_-_-_-_-_-_-_-_-COO

K

F - A - M - K

K - F - A - M

Q - M - K

D - I - K - Q - M

G - M - D - I - K

Y - R - G - M

Y - R

Cyanogen Bromide (CN Br)

Cleaves after Met i.e M - X

D - I - K - Q - M

K

K - F - A - M

Y - R - G - M

Trypsin cleaves after K or R

(positively charged amino acids)

Q - M - K

G - M - D - I - K

F - A - M - K

Y - R

33

For Next Time:

Finish Chapter 4! We are moving on to

Chapter 5.

Finish Chapter 4 Problems

Start Chapter 5 Problems