-helix right-handed 3.6 aa per turn hydrogen bond n-h···o=c collagen triple helix: three...

-helix• right-handed• 3.6 aa per turn• hydrogen bond N-H···O=C

Collagen triple helix: three polypeptide

intertwined

A stereo, space-filling representation

B2 Protein structure － Secondary

-sheet: hydrogen bonding of the peptide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chainx

A stereo, space-filling representation of the six-stranded antiparallel sheet.

Parallel sheet: sections run in the same directionAntiparallel sheet: sections run in the opposite direction

fibroin

A two-stranded sheet

B2 Protein structure and function

B2 Protein structure － TertiaryThe different sections of -helix, -sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions

Noncovalent interaction between side chains that hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactionsCovalent interaction: disulfide bonds

Denaturation of protein by disruption of its 2o and 3o structure will lead to a random coil conformation

B2 Protein structure and function

Many proteins are composed of two or more polypeptide chains (subunits). These subunits may be identical or different. The same forces which stabilize tertiary structure hold these subunits together. This level of organization called quaternary structure.

A stereo, space-filling drawing showing the quaternary structure of hemoglobin

1-yellow; 1-light blue; 2-green; -dark blue; heme-redback

B2 Protein structure － Quaternary

Advantages of the quaternary structure:1. It allows very large protein molecules to be mad

e, such as tubulin 。2. It can provide greater functionality to a protein b

y combining different activities into a single entity.

3. The interactions between the subunits can often be modified by binding of small molecules and lead to the allosteric effects seen in enzyme regulation

Protein Folding: chaperones are involved in vivo

(1) The rapidly reversible formation of local secondary structure

(2) Formation of domains through the cooperative aggregation of folding nuclei

(3) Assembly of domains into a “molten” globule

(4) Conformational adjustment of the monomer

(5) Final conformational adjustment of the dimeric protein to form the native structure.

back

B2 Protein structure and function

Prosthetic groups ( 辅基 ): covalently or noncovalently attached to many conjugated proteins, and give the proteins chemical functionality. Many are co-factors in enzyme reactions. Apoprotein ( 脱辅基蛋白 )Examples : heme groups in hemogobin (Figure)

B2 Protein structure and function

Biological functions of proteins

•Enzymes: substrate binding, side chain in catalysis

•Signaling: cell membrane

•Transport and storage: hemoglobin transports oxyge

n

•Structure and movement: collagen, keratin, tubulin i

n cytoskeleton, actin and myosin for muscle contractio

n

•Nutrition: casein ( 酪蛋白 ) and ovalbumin( 卵清蛋白 )

•Immunity: antibodies

•Regulation: transcription factors

B2 Protein structure and function

B2 Protein structure － Domains, motifs and families

Domains: structurally independent units of many proteins, connected by sections with limited higher order structure within the same polypeptide. (Figure)

They can also have specific function such as substrate binding

Structural motifs: • Groupings of secondary structural elements that frequently occur in globular proteins• Often have functional significance and represent the essential parts of binding or catalytic sites conserved among a protein family

motif

Protein families: structurally and functionally related proteins from different sources

The primary structures of c-type cytochromes from different organisms

Motif

Protein purification

• An experimental step to purify the interested protein from other proteins and nonprotein molecules existing in the cells

• An essential experimental step when study any individual protein

The principal properties of proteins used for purification

1. Size: gel filtration chromatography

2. Charge: ion-exchange chromatography, isoelectric focusing electrophoresis

3. Hydrophobicity: hydrophobic interaction chromatography

4. Affinity: affinity chromatography

5. Recombinant techniques: involving DNA manipulation and making protein purification so easy

1. gel filtration chromatography

2. Charge: ion-exchange chromatography, isoelectric focusing, electrophoresis

Isoelectric point (pI): the pH at which the net surface charge of a protein is zero

--

--

-- -

-

++

+

+

+

+ ++

pH=pIpH>pI pH<pI

Ion-exchange chromatography

Column + anions

+++

Sample mixture

Protein binding

Column + proteins Column + anions

Ion displacing

Purified protein

+

Electrophoresis

Protein migrate at different position depending on their net charge

Isoelectric focusing

A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel.

3. Hydrophobicity: hydrophobic interaction chromatography

Similar to ion-exchange chromatography except that column material contains aromatic or aliphatic alkyl groups

4. Affinity chromatography

ding

d

• Enzyme-substrate binding

• Receptor-ligand binding

• Antibody-antigen binding

Substrate analogs: competitive inhibitors

5. Recombinant techniques:

•Clone the protein encoding gene of interest in an expression vector with a purification tag added at the 5’- or 3’ end of the gene

•Protein overexpression in a cell

•Protein purification with affinity chromatography.

Mass Determination

Gel filtration chromatography and SDS-PAGE

•Comparing of the unknown protein with a proper standard

•Popular SDS-PAGE: cheap and easy with a 5-10% error

•SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass.

Mass Determination

Mass spectrometry:

• Molecules are vaporized and ionized (by Xe/Ar beam), and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured

• Extremely accurate (0.01% error), but expensive

• ESI (electrospray ionization) and MALDI (matrix-assisted laser desorption/ionization) can measure the mass of proteins smaller than 100 KDa

• Helpful to detect post-translational modification

• Protein sequencing: relying on the protein data base

Determine the primary structure of a protein: p

Amino acid composition:

1. Acid treatment to hydrolyze peptide bonds: 6M HCl, 110°C for 24 hrs.

2. Chromatographic analysis

However, you cannot get the sequence!

Specific enzyme/chemical cleavage: •Pepsin cleaves after lysine(K) or arginine (R)•V8 protease cleaves after glutamic acid (E)• Cyanogen bromide cleaves after methionine (M)

Edman degradation:•Performed in an automated protein sequencer•Determine the sequence of a polypeptide from N-terminal amino acid one by one. Phenylisothiocyanate 苯异硫氰酸 reaction)•Expensive and laborious

Determine the primary structure of a protein: protein sequencing

Protein sequence analysis (1)

Sequence: HLMGSHLVDALELVMGDRGFEYTPKAWLV

Pepsin T1 HLMGSHLVDALELVMGDR

T2 GFEYTPK

T3 AWLV

V8 V1 HLMGSHLVDALE

V2 LVMGDRGFE

V3 YTPKAWLV

Protein sequence analysis (2)

Computer analysis:

T1 HLMGSHLVDALELVMGDR

V1 HLMGSHLVDALE

V2 LVMGDRGFE

T2 GFEYTPK

V3 YTPKAWLV

T3 AWLV

Sequence: HLMGSHLVDALELVMGDRGFEYTPKAWLV

Most protein sequences are deduced from the DNA/cDNA sequence

Direct sequencing: determine the N-terminal sequences or some limited internal sequence construction of an oligonulceotide or antibody probe fishing the gene or cDNA

X-ray crystallography and NMRDeterming the 3-D structure of a protein

X-ray crystallography:

• Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first hand data obtained is electron density map, the crystal structure is then deduced.

• A very powerful tool in understanding protein 3-D structure

• Many proteins have been crystallized and analyzed

X-ray crystallography and NMRDeterming the 3-D structure of a protein

• Measuring the relaxation of protons after they have been excited by radio frequencies in a strong magnetic field

• Measure protein structure in liquid but not in crystal

• Protein measured can not be larger than 30 KDa

NMR: Nuclear magnetic resonance (NMR) spectroscopy

Summary

1. 20 Amino acids: D & E (-), H, K, R (+), S, T, C, N, Q (polar), G,A,V,L, I, M, P (nonpolar), F, Y,W (aromatic)

2. Protein structure: primary, secondary, tertiary, quaternary

3. Protein analysis: purification, sequencing, mass determination, X-ray crystallography and NMR