OD 660

請尊重與保護智慧財產權 僅提供你個人使用

Chapter 3 (p52)

Amino Acids and the

Primary Structure of

Proteins

Important biological functions of proteins (p53)

1. Enzymes, the biochemical catalysts 2. Storage and transport of biochemical

molecules

3. Physical cell support and shape (tubulin, actin, collagen)

4. Mechanical movement (flagella, mitosis,

muscles)

Important biological functions of proteins (p53)

5. Decoding cell information (translation, regulation of gene expression)

6. Hormones or hormone receptors (regulation of cellular processes)

7. Other specialized functions (antibodies,

toxins etc.)

3.1 (p53) General structure of amino acids

• 20 common α-amino acids

• Carboxyl and amino groups bonded to the ^α -carbon atom

• A hydrogen atom and a side chain (R) are also attached to the α-carbon atom

Zwitterionic form of amino acids (p54)

• Under normal cellular conditions

amino acids are zwitterions (dipolar ions):

Amino group = -NH

Carboxyl group = -COO

Fig 3.1 (p54) Two representations of an amino acid at neutral pH

Stereochemistry (p54)

• Stereoisomers

- the same molecular formula

- differ in the arrangement of atoms in space

• Enantiomers- nonsuperimposable mirror images

• Chiral carbons- have four different groups

attached

Stereochemistry of amino acids ^(p54)

• 19 of the 20 common amino acids have a chiral α- carbon atom (Gly does not)

• Threonine and isoleucine have 2 chiral carbons each (4 possible stereoisomers each)

• Mirror image pairs of amino acids are designated L (levo) and D (dextro)

• Proteins are assembled from L-amino acids

(a few D-amino acids occur in in the cell walls of certain microorganism)

D, L System (p55)

•Absolute configurations of all carbon-

based molecules are referenced to D- and L-glyceraldehyde.

•Asymmetric carbon most remote from the aldehyde or the ketone end of the

molecule related to the asymmetry

carbon of glyceraldehyde.

D, L System

• R-CHX-R’ R’: longer carbon chain R’: bottom

R: top

R R

⎜ ⎜

H-C-X X-C-H

⎜ ⎜

R’ R’

D-form

L-form

Fig 3.2 (p55) Mirror-image pairs of amino

acids

Assignment of configuration by the R, S System (p56)

* View the molecule from the chiral center to the atom with the lowest priority

• R configuration

– The other three atom facing the viewer decrease in priority in a clockwise direction

• L configuration

– The three atoms decrease in priority in a counterclockwise direction

Assign a priority (p56)

(a) Based on atomic mass priority

- Each group attached to a chiral carbon

- If two atoms are identical, move to the next atoms - Highest, 1

- Lowest, 4

• For double or triple bonds, count atom once for each bond (-CHO higher priority than -CH₂OH)

• Priorities (low to high): -H, -CH₃, -C₆H₅, -CH₂OH, -CHO, -COOH, -NH₂, -NHR, -OH, -OR, -SH

RS system (p56)

(b) Orient the molecule with priority 4 pointing away (behind the chiral carbon). Trace path from highest priority to lowest priority (1, 2, 3, 4)

(c) Clockwise path: absolute configuration R

Counterclockwise path: absolute configuration S NOTE: All of the 19 common chiral L-amino acids

except cysteine have the S configuration.

Assignment of RS configuration (p56)

d and l system

• d: (+), detrorotatory, clockwise rotation

• l: (-), levorotatory, counter clockwise rotation

• Optical active compound

• Rotation of plane-polarized light

• Plane-polarized light

light whose vibrations take place in only

one plane

3.2 (p55) Structures of the 20 common amino acids

• Fischer projections

- horizontal bonds from a chiral center extend toward the viewer

- vertical bonds extend away from the viewer

• Abbreviations can be one letter or three letters

• Amino acids are grouped by the properties of their side chains (R groups)

• Classes: Aliphatic, Aromatic, Sulfur-containing, Alcohols, Bases, Acids and Amides

P57 Box 3.2

A. Aliphatic R Groups

• Glycine (Gly, G) - the α-carbon is not chiral since there are two H’s attached (R=H)

• Four amino acids have saturated side chains:

Alanine (Ala, A) Valine (Val, V) Leucine (Leu, L) Isoleucine (Ile, I)

• Proline (Pro, P) 3-carbon chain connects α-C and N

Aliphatic amino acid structures ^(p57)

Ile

Fig 3.3 Stereoisomers of Isoleucine

• Ile has 2 chiral carbons, 4 possible stereoisomers

Proline has a nitrogen in the aliphatic ring system

• Proline (Pro, P) - has a three carbon side chain bonded to the α-amino nitrogen

• The heterocyclic pyrrolidine ring restricts the geometry of

polypeptides.

B. Aromatic R Groups

• Side chains have aromatic groups

Phenylalanine (Phe, F)- benzene ring Tyrosine (Tyr, Y)- phenol ring

Tryptophan (Trp, W)-.bicyclic indole group

Aromatic amino acid structures

C. Sulfur-Containing R Groups

• Methionine (Met, M) - (-CH₂CH₂SCH₃)

• Cysteine (Cys, C) - (-CH₂SH)

• Two cysteine side chains can be cross-linked by forming a disulfide bridge (-CH₂-S-S-CH₂-)

• Disulfide bridges may stabilize the three- dimensional structures of proteins.

Methionine and cysteine (p59)

Fig 3.4 (p59) Formation of cystine

D. Side chains with alcohol groups (p59)

• Serine (Ser, S) and Threonine (Thr, T) have uncharged polar side chains

E. Basic R Groups

• Histidine (His, H)- imidazole

• Lysine (Lys, K)- alkylamino group

• Arginine (Arg, R)- guanidino group

• Side chains are nitrogenous bases which are substantially positively charged at pH 7.

Structures of histidine, lysine and arginine

(p60)

F. Acidic R groups and amide derivatives (p60)

• Aspartate (Asp, D) and Glutamate (Glu, E)

• dicarboxylic acids

• negatively charged at pH 7

• Asparagine (Asn, N) and Glutamine (Gln, Q) are uncharged but highly polar.

Structures of aspartate, glutamate,

asparagine and glutamine (p60)

G. Hydrophobicity of amino acid side chains (p60-61)

• Hydropathy: the relative hydrophobicity of each amino acid

• The larger the hydropathy, the greater the tendency of an amino acid to prefer a hydrophobic environment

• Hydropathy affects protein folding:

hydrophobic side chains tend to be in the interior.

hydrophilic residues tend to be on the surface.

Table 3.1 (p60)

• Hydropathy scale for amino acid residues

3.3 Other amino acids and amino acid derivatives (p61)

• Over 200 different amino acids are found in nature.

• Most are precursors to common amino acids or chemically modified derivatives.

• Some amino acids are chemically modified after incorporation into a polypeptide.

Fig 3.5 (p61) Compounds derived from

common amino acids

21st Amino acid- Selenocysteine (p62)

• Selenocysteine is

incorporated into a few proteins.

• Constitutes the 21^st amino acid

• Have its own codon.

22nd amino acid-Pyrrolysine (p62)

• Modified form of Lys

• Have its own codon

3.4 Ionization of amino acids (p62)

• Ionizable groups in amino acids: (1) α-carboxyl, (2) α -amino, (3) some side chains

• Each ionizable group has a specific pK_a

AH B + H⁺

• pH below the pK_a, the protonated form predominates (AH)

• pH above the pK_a, the unprotonated form predominates (B)

Fig 3.6 Titration curve for alanine

Fig 3.7 (p64) Ionization of Histidine

(p63)

Table 3.2 (p64)

pK_a values of amino acid ionizable groups

Henderson-Hasselbach equation:

calculating group ionizations (p64)

(p64)

Fig 3.8 (p65)

(b) Deprotonation of the guanidinium group of Arg

(a) Ionization of the protonated

3.5 Peptide bonds link amino acids in proteins (p66)

• Peptide bond- linkage between amino acids, a secondary amide bond

• Condensation of the α-carboxyl of one amino acid with the ^α-amino of another amino acid (loss of

H₂O molecule)

• Primary structure- linear sequence of amino acids in a polypeptide or protein

Fig 3.9 Peptide bond between

two amino acids (p66)

Polypeptide chain nomenclature (p67)

• Amino acid “residues” compose peptide chains

• Numbered from the N (amino) terminus to the C (carboxyl) terminus

• Example: (N) Gly-Arg-Phe-Ala-Lys (C) (GRFAK)

• Formation of peptide bonds eliminates the ionizable α- carboxyl and α-amino groups of the free amino acids

Fig 3.10 Aspartame, an artificial sweetener (p67)

• A dipeptide methyl ester (aspartylphenylalanine methyl ester)

• About 200 times sweeter than table sugar

• Used in diet drinks

Reaction of Amino Acids

primary amine

alcohol

aldehyde

acid halide acid anhydride

Ninhydrin reaction

• Ninhydrin is a strong oxidant and causes the oxidative deamination of the α-amino

group.

• Typical reaction products of α-amino acid absorb light at 570 nm.

• α-Imino acids, such as proline and

hydroxyproline, give bright yellow products

with absorption maxima at 440 nm

Ninhydrin reaction

oxidative deamination

Light → Molecule Ê

Ì Absorption by chromophore

È

Excitation energy

Á Ì

Energy

(Heat)

Absorption

• I = I₀10^-εdc

• I = Intensity of the transmitted light

• I

= Light intensity

• ε = Molar absorption coefficient or molar extinction coefficient

Absorption of a 1 M solution of pure compound under standard condition

• d = thickness in cm

• c = molar concentration

• log₁₀ (I/ I₀) = -εdc ----Beer-Lambert law

% Transmission = (I/ I

) X 100 Absorbance A = -log (I/ I

)

or A = log (I

/I)

• When d = 1 cm

A is called OD (Optical Density)

• A = εdc

1.37 X 10^-4 M NADH , OD₃₄₀ = 0.85, calculate the molar extinction coefficient

• A = εdc

• ε = A/dc = 0.85/(1 cm x 1.37 x 10

moles/l)

= 6.20 X 10

cm

/mole or M

cm

• 1 liter = 10

cm

Spectroscopic properties of amino acids

• Ultraviolet (UV) spectra

• Aromatic amino acids, phenylalanine,

tyrosine, and tryptophan absorb above 250 nm.

• All amino acids absorb in the infrared

region.

Ultraviolet spectra of aromatic amino acids

Estimation of protein concentration in solution

• Lowry method

• BCA method

• Bradford assay

Lowry method

• Use a Cu

and Folin-Ciocalteau reagent (a combination of phosphomolybdic and

phosphotungstic acid complex)

• Cu⁺ is generated

from Cu

by oxidizable

protein components, such as cysteine or the phenols and indoles of tyrosine and

tryptophan

• The Cu

reaction with the Folin reagent

gives blue color products

BCA method

• BCA (Bicinchoninic acid) forms a

purple complex with Cu

in alkaline

solution.

BCA method

Bradford assay

• Coomassie dye binding protein assay

• Coomassie Brillant Blue G

• Reactive to Arg and Lys

• Reaction product: OD

Standard curve

• Standard protein

• BSA (Bovine Serum Albumin)

• BSA, μg OD₆₆₀ by Lowry method

0 0

1 0.1

5 0.2

10 0.5

15 0.75

Sample 0.3

20 18

16 14

12 10

8 6

4 2

0 0.102 0.092 0.082 0.072 0.061 0.051 0.041 0.031 0.02 0.01 0

r = 1.000

BSA, μ g OD 660

20 18

16 14

12 10

8 6

4 2

0 0.246 0.225 0.205 0.184 0.164 0.143 0.123 0.102 0.082 0.061 0.041 0.02 0

BSA, μ g OD 660

r = 0.9975

請尊重與保護智慧財產權 僅提供你個人使用