Chapter 2Chapter 2

Structures and Functions of Protein

Functions of ProteinA. Structural support: providing matrix, giving form to liv

ing organism, serving as structural components of cells.

B. Catalysis: enzymes catalyze chemical reaction

C. Transport: oxygen, iron, lipid, etc.

D. Coordinated motion: myosin and actin in muscle contraction

E. Specific binding to molecules Immune protection: immunoglobulin(Ig), interferon. Regulation of metabolism: insulin, growth hormone Regulation of gene expression: transcription factor

What is protein ?

Proteins are polypeptides, which are linear polymers of amino acids linked together by peptide bonds.

Proteins are synthesized from 20 different amino acids.

Quantification of proteins

Proteins are the major compounds containing nitrogen, and with the average of 16% of the molecular weight, proteins can be quantified by measuring the amount of nitrogen.

1 gram of nitrogen equals to 6.25 gram of protein

Amino Acids: there are 20 standard amino acids that make up almost all naturally existing proteins.

Amino acid structures can be displayed as:

Structure of amino acids

H

glycine

CH3

alanineL-amino acidgeneral structure

R C +NH3

COO-

H

Mirror image structure of amino acids

D : dextrorotation L:laevogyrate

(D) COOH or (L) COOH

H C H2N H2N C H

R R (Only L-amino acids are found in proteins)

20 Standard Amino Acids

Name 3-letter 1-letter Name 3-letter 1-letterAlanine Ala A Leucine Leu LArginine Arg R Lysine Lys KAsparagine Asn N Methionine Met MAspartate Asp D Phenylalanine Phe FCysteine Cys C Proline Pro PGlutamine Gln Q Serine Ser SGlutamic acids Glu E Threonine Thr TGlycine Gly G Tryptophan Trp WHistidine His H Tyrosine Tyr YIsoleucine Ile I Valine Val V

Essential Amino Acids

There are 8 amino acids that can not be adequately synthesized by the body and therefore must be obtained from the diet. These amino acids are called essential amino acids, otherwise, called nonessential amino acids. Essential amino acids include:

Lys, Trp, Val, Leu, Ile, Thr, Met, Phe.

Structural characteristics of Amino Acids

1) each has an -amino group, an -carboxyl group, and a side-chain (except Gly). Amino acids are different in their side-chains (or R-groups).

2) each has two enantiomers due to the asymmetric -carbon atom (except Gly), but in proteins there are only L-amino acids.

3) the groups bounded to the -carbon can rotate to give different conformations (except Pro).

Classification of Amino Acids

According to the polarities of their R-groups,

amino acids can be classified as:

1) Neutral non-polar AA : Gly, Ala, Val, Leu, Ile, Phe, Pro.(with non-polar aliphatic or aromatic side chains )

2) Neutral polar AA: Trp, Ser, Tyr, Cys, Met, Asn, Gln,

Thr. (with polar non-ionic side chains )

3) Acidic AA: Asp, Glu. (with ionic side chains )

4) Basic AA: Arg, Lys, His. (with ionic side chains )

甘氨酸 glycine Gly G 5.97

丙氨酸 alanine Ala A 6.00

缬氨酸 valine Val V 5.96

亮氨酸 leucine Leu L 5.98

异亮氨酸 isoleucine Ile I 6.02

苯丙氨酸 phenylalanine Phe F 5.48

脯氨酸 proline Pro P 6.30

1.Neutral non-polar AA. Structure Name Name 3-letter 1-letter pI

色氨酸 tryptophan Try W 5.89

丝氨酸 serine Ser S 5.68

酪氨酸 tyrosine Try Y 5.66

半胱氨酸 cysteine Cys C 5.07

蛋氨酸 methionine Met M 5.74

天冬酰胺 asparagine Asn N 5.41

谷氨酰胺 glutamine Gln Q 5.65

苏氨酸 threonine Thr T 5.60

2. Neutral polar AA

天冬氨酸 aspartic acid Asp D 2.97

谷氨酸 glutamic acid Glu E 3.22

赖氨酸 lysine Lys K 9.74

精氨酸 arginine Arg R 10.76

组氨酸 histidine His H 7.59

3. Acidic AA

4. Basic AA

-OOC-CH-CH2-S+NH3

S-CH2-CH-COO-

+NH3

-OOC-CH-CH2-S+NH3

S-CH2-CH-COO-

+NH3

Cysteine

+

CystineDisulfide bond

-HH

-OOC-CH-CH2-SH

+NH3

-OOC-CH-CH2-SH

+NH3

HS-CH2-CH-COO-

+NH3

HS-CH2-CH-COO-

+NH3

Special amino acids

Proline（ imino acid)

CH2

CHCOO-

NH2+

CH2

CH2

CH2

CHCOO-

NH2+

CH2

CH2

Physicochemical Properties of Amino Acids

1) Isoelectric point: at physiological pH, amino acids exist as dipolar ion (zwitterion).

R-CH-COOH

NH2

R-CH-COOH R-CH-COO- R-CH-COO-

NH3+

NH3 + NH2

Cation Zwitterion Anion

+OH- +OH-

+H++H+

Therefore, the isoelectric point (pI) of an amino acid is the pH at which it has no net charge.

pI depends on dissociation constants of α-NH

3+ and α-COOH of an amino acid.

When pH=pI, net charge=0 (zwitterion);

When pH>pI, net charge<0 (negative);

When pH<pI, net charge>0 (positive).

Roughly calculate the pI for an amino acid:

A) If:

K1 K2

+H3N-R-COOH +H3N-R-COO- H2N-R-COO-

then: pI= (pK1+ pK2)/2

2) Ultraviolet light absorption: some amino acids such as Trp(tryptophan ) and Tyr (tyrosine ) have a peak absorption at a wavelength of 280nm. This property is commonly used to determine the concentration of proteins in solution.

3) Ninhydrin reaction: Ninhydrin + amino acid blue-purple compound

This reaction is also used to quantitatively determine amino acids and proteins.

2. Peptide1) Peptide bond: a covalent bond between

the -amino group of one amino acid and the -carboxyl group of another.

H2N-CH-COOH + H2N-CH-COOH

R1 R2

H2O

H2N-CH-CO HN-CH-COOH

R1 R2

NH2-CH-CH OH

NH -O

2 CH-CH OH

glycine

+

-HOH

GlycylglycinePeptide

bond

NH2-CH-C-N-CH-CO

OHHHH

ONH2-CH-C-N-CH-C

O

OHHHH

O

glycine

NH CH COO

- -H OHH

2) Peptide: a linear structure formed by two or more amino acids linked via peptide bonds.

e.g. dipeptide, tripeptide, and octapeptide, are formed from two, three, and eight amino acids, respectively.

Generally, a peptide containing 2-10 amino acids is called oligopeptide; more than 10 amino acids form a longer peptide called polypeptide.

Amino acids in a peptide are called amino acid residues.

3. Molecular structure of proteins

Primary structure

Secondary structure

Tertiary structure

Quaternary structure

HigherStructures

Primary structure of proteins

1) Primary structure: refers to the linear sequence of amino acids in a protein.

1) The major linkage of the primary structure is peptide bond. In some cases disulfide bond is also involved.

3) Spatial structure and function of a protein depend on its primary structure.

Note: primary structure is not the only factor to

maintain the spatial structure of a protein.

4) If the primary structure is altered, the function of the protein may also be changed.

e.g. bovine insulin, the first protein whose complete

amino acid sequence was determined by Frederick Sanger in 1953.

Primary structure of bovine insulin

disulfide bond

Secondary structure1) Secondary structure of protein refers to the

conformation adopted by local regions of polypeptide chain, just refers to the spatial conformation of the main chain of a peptide, regardless of the distribution of side chains.

Secondary structures include: -helix, -pleated sheet, β-turn, etc. The irregular secondary structures are termed random coil or loop.

2) The force to maintain the secondary

structure of a protein is hydrogen bond.

-helixes: include right- and left-handed -

helixes.

Peptide unit

• Peptide unit: peptide unit is a rigid , planar structure. The 4 members of the amide bond and other 2 α-carbons, which connect to the carboxyl carbon and the amino nitrogen respectively , are on the plane. This plane is termed peptide unit .

Amide Planes

Properties of peptide unit: The peptide’s C-N bond is 0.132nm.

N-Cα single bond is 0.147nm.

With ~40% double bond character, the amide C-N bonds are unable to rotate freely.

1)The six atoms of the peptide unit lie in a single plane. H and O are trans to each other.

2)The Cα-C bond and the N-Cαbonds are common

single bonds and permitted to rotate. Ψ(psi) for the Cα-C

bond and φ (phi) for the N-Cα bond,

Left- and right-handed -helixes

Right-handed -helix

left-handed -helix

-helix

0.54nm(3.6 residues)

Characters of-helix

α-helical structure is the simplest arrangement with its rigid peptide bonds

1) the peptide backbone is tightly wound around the long

axis of the molecule, and the helix is right handed with

fixed torsion angles ( = -57 = -47

2) At this right handed helix, 3.6 amino acid residues per

turn, and a pith of 5.4 nm are found. This means that the

span between two neighboring amino acids is 0.15 nm.

.

3) The hydrogen bonds are the major bonds to stabilize the helix . The peptide C=O bond of a peptide unit points along the helix towards N-H group of the 4th peptide unit to form a hydrogen bonds . Each hydrogen bond closes the peptide chain to form a ring with 13 atoms.

4) R groups of the amino acid residues protrude outward from the helical backbone.

-pleated sheet1)The angles between peptide units are 110°, and the backbone of t

he polypeptide chain is extended into a zigzag shape.

2)The zigzag polypeptide chains are arranged side by side to form a series of pleats.

3)In a -pleated sheet, the hydrogen bond occurs between neighboring segments of polypeptide chains.

4)The neighboring hydrogen bonded polypeptide chains run in the opposite directions (antiparallel) or in the same direction (parallel)

5) R groups of adjacent amino acids protrude in opposite directions from the zigzag structure.

-pleated sheets:

hydrogen bonding

Peptide 1

Peptide 2

110 °

anti-parallel  N C     parallel  N C                    C N                  N C

-pleated sheets: include parallel (running in the same direction, ) and antiparallel (running in opposite directions) -pleated sheets.

The -pleated sheet

R groups of adjacent amino acids protrude in opposite directions from the zigzag structure.

-turn: -turn is a reverse turn and often occurs at protein surfaces. Most-turns involve four successive amino acid residues and stabilized by hydrogen bonds located between residue 1 and 4.

-turn is formed when the polypeptide chain folds to make a 180-degree turn.

• Motif: certain arrangements of several (mostly 2-3) secondary structure elements with some specific functions can occur to form different protein structures, named motifs.

• They include the helix-turn-helix motif, zinc finger motif , etc.

(zinc finger)(zinc finger)

N-terminal

C- terminal

Tertiary structure: the overall folding of a Poly-peptide chain including the spatial distribution of all atoms in the molecule. This refers to the spatial relationships between secondary structure elements. e.g. myoglobin:

The forces to maintain the tertiary structure of

a protein include hydrogen bond, hydrophobic

interaction, salt bonds, Van Der Waals force.

Generally, the hydrophobic side-chains are

buried in the interior and the polar or charged

side-chains are on the surface of the protein.

Forces to maintaining spatial conformation a hydrogen bonds ， b salt bonds c hydrophobic interaction

Quaternary structure: refers to the spatial arrangement of subunits in a multisubunit protein.

A protein with quaternary structure contains at least two polypeptides (or subunits).

The forces to maintain the quaternary structure of a protein mainly are hydrogen bond, salt bond, Van Der Waals force and hydrophobic interaction

(Hemoglobin)

primary linear sequence (peptide bond) 1D

secondary local region, spatial arrangements 2-D

(hydrogen bonds)

tertiary three dimensional structure of overall fold  3-D

(hydrogen bond, salt bond, Van Der Waals force and hydrophobic interaction)

quaternary non-covalent multimerization of subunits 4-D

(single polypeptides) into protein complexes  (hydrogen bond, salt bond, Van Der Waals force and hydrophobic interaction)

Summary of Structure of Proteins

Primary structure dictates folding of higher structures

Primary structure-function relationship of Protein

• The primary structure of a protein is the material base of its three-dimensional structure.

• The spatial structure of a protein determines its biological function.

• Proteins have similar amino acid sequences will have similar spatial structures and functions.

Primary structure-function relationships of InsulinIsulins from different animal species contain two polypeptide chains and have high homogeneity and similar three dimensional structure. Isulins from different animal species show a residue identity at 22 amino acid positions.

There are great varieties in A5, A6, A10 and B30, but the biological function is identical.

4 amino acid residues are essential for maintaining the the correct spatial structure and function (Gly-Ile-Val-Gl

u.)

Variation in positions in insulines from some animal species

Species Variation in positions in insulines from some animal species

A5 A6 A10 B30

Human Thr Ser Ile Thr

Pig Thr Ser Ile Ala

Dog Thr Ser Ile Ala

Rabbit Thr Ser Ile Ser

Cow Ala Ser Val Ala

Sheep Ala Gly Val Ala

Horse Thr Gly Ile Ala

Sperm whale Thr Ser Ile Ala

N-val · his · leu · thr · pro · glu · glu · · · · ·C(146)

HbS β chain

HbA β chain

N-val · his · leu · thr · pro · val · glu · · · · ·C(146)

Sickle cell hemoglobin: The hydrophilic residue glutamic acid at 6th position of β chain is substituted by a hydrophobic residue valine. This results into a high rate of hemolysis and sickle cell anemia.

This kind of disease caused by site mutation of gene coding this protein is termed “molecular disease”

Sickle cell

Structure-function relationship of myoglobin and hemoglobin

Myoglobin (Mb): the first protein to have its 3-d

structure solved by X-ray crystallography. It is located

in muscle.

The major function of Mb is bind, store, and release O2

with changes in the O2 concentration in skeletal muscle.

Mb + O2 MbO2

Structural characteristics of myoglobin: a) it consists of a single polypeptide chain of 1

53 amino acid residues and a heme prosthetic group;

b) it has a compact structure with 75% of the residues in -helix, referred to as “A” through “H”;

c) Hydrophobic groups locate in the core of the molecules, hydrophilic groups expose at the surface of the spherical molecule.

Myoglobin

•

N-terminal

C- terminal

Tertiary structure of myoglobin

F8 His

Porphyrinplane

Structure of heme

Hemoglobin (Hb): hemoglobin is a tetrameric

protein which consists of two -chains (141

residues each) and two -chains (146 residues

each). The conformation of each subunit is

similar to that of myoglobin.

Function: as the oxygen transporter in RBC.

Hb + 4O2 Hb(O2)4

                                                                

Salt bonds between and within subunits in deoxyHb

Conformations of hemoglobin

Oxygen dissociation curves of Hb and Mb

This shape indicates that the affinity of Hb for binding

o2 is relatively low.

Cooperativity

Cooperativity refers to the allosteric effect of

the action of one subunit on other’s biological

activity. If the action promotes the activity of

other subunits, it is said to be positive; if

otherwise inhibits other subunit’s activity,

then negative co-operativity.

O2

F8 His

F8 His

F peptid

F peptid

Porphyrin plane

Allosteric property of hemoglobin

Hb is an allosteric protein: binding of O2 to one

subunit induces conformational change of other

subunits, making them more able to bind O2.

O2 (causing the changes) is nanmed allosteric

Effecter.

O 2 O 2 O 2 O 2 O 2 O 2 O 2

O 2 O 2 O 2

Protein conformational disease Mad cow disease is an illness that attacks the

brain and spinal cord in adult cattle, caused by p

rion protein (PrP) PrPsc

PrPc

α-helix

PrPsc

β-pleated sheet

normal Mad cow disease

Prion protein(PrPsc)

Prion protein(PrPsc)

Prion disease involves a novel mechanism of pathology which is the alteration of the secondary – tertiary structure of a naturally occurring protein, PrPc.

Physicochemical properties of protein

1) Isoelectric point(pI): proteins contain ionizable groups such as amino and carboxyl groups. When the net charge is zero the pH is referred to as pI of the protein.

NH3+ NH3

+ NH2

P P P

COOH COO - COO -

Cation(pH<pI) Zwitterion (pH=pI) Anion (pH>pI)

+OH- +OH-

+H+ +H+

2) Denaturation, precipitation and coagulation:Denaturation refers to the loss of native confor

mation and biological activity of the protein. Denatured proteins decrease in solubility, increase in stickiness, and are vulnerable to proteolytic enzymes.

Precipitation is the process by which protein separates out from the aqueous solution. Protein can be precipitated by removal of the two stabilizing factors: electrostatic repulsion and hydration shell.

Coagulation is the solidification change of proteins caused by physical or chemical factors.

3) Ultraviolet light absorption of proteins results from the residues that have peak absorbance at 280nm such as Trp, Tyr. This property is commonly used for quantitative determination of proteins.

4) Color reactions: such as ninhydrin and biuret reactions. Color reactions are also used for analysis of proteins.