1. Section B Protein Structure
Molecular Biology Course
Section B
Protein Structure

2. B2 Protein structure and function:
Cross with Biochemistry
Molecular Biology Course
B1 Amino acids: structure, side chains (charged, polar uncharged, nonpolar aliphatic, aromatic)
B2 Protein structure and function:
Structure: size and shapes, primary secondary  tertiary  quaternary (prosthetic groups) ; Biological functions
Structure & function: Domains, motif, and family
B3 Protein analysis
Purification  Determine sequence, mass, and
structure (X-ray crystallography and NMR)

3. B1 Amino acids- basic structure
Protein structure
B1 Amino acids- basic structure
H3N
COO- R H Ca
Common structure of 19 AAs
proline
a-carbon is chiral (asymmetric) except in glycine (R is H)
2. Amino acids are dipolar ions (zwitterions [兼性离子]) in aqueous solution and are amphoteric (兼性的同时有酸碱性或正负电荷的)
3. The side chains (R) differ in size, shape, charge and chemical reactivity
4. A few proteins contain nonstandard amino acids that are formed by post-translational modification of proline and lysine.

4. B1 Amino acids- charged (5)
Protein structure
B1 Amino acids- charged (5)
Form salt bridges, are hydrophilic (亲水)
1. “Acidic” amino acids (2): containing additional carboxyl groups which are usually ionized
aspartic acid (Asp, D，天冬氨酸)
glutamic acid (Glu, E，谷氨酸)

5. Lysine (Lys, K, 赖氨酸) Arginine (arg, R,精氨酸) Histidine (His, H，组氨酸)
Protein structure
2. “Basic” amino acids (3): containing positively charged groups e
Lysine (Lys, K, 赖氨酸)
Arginine (arg, R,精氨酸) d
a guanidino group (胍基）
Histidine (His, H，组氨酸)
The imidazole group (咪唑基) has a pKa
near neutrality. This group can be reversibly
protonated under physiological conditions,
which contribute to the catalytic mechanism of many enzymes.

6. Contain groups that form hydrogen bonds with water,
Protein structure
B1 Amino acids- polar uncharged (5)
Contain groups that form hydrogen bonds with water,
hydrophilic
Serine (Ser, S，丝氨酸)
Contain hydroxyl groups.
Threonine (Thr, T，苏氨酸)
Asparagine (Asn, N，天冬酰氨)
Glutamine (Gln, Q，谷氨酰氨)

7. Protein structure
Cysteine (Cys, C，半胱氨酸) has a thiol (巯醇) group， which is often oxidizes to cystine
x-S-S-x

8. B1 Amino acids- nonpolar aliphatic (7)
Protein structure
B1 Amino acids- nonpolar aliphatic (7)
(hydrophobic 疏水)
Glycine (Gly, G，甘氨酸)
Proline (Pro, P，脯氨酸): imino acid (亚氨基酸)
Methionine (Met, M，甲硫氨酸): contains a sulfur atom

9. Isoleucine (Ile, I，异亮氨酸)
Protein structure
Alkyl (烷基) side chains
Alanine (Ala, A，丙氨酸)
Leucine (Leu, L，亮氨酸)
Valine (Val, V，缬氨酸)
Isoleucine (Ile, I，异亮氨酸)

10. B1 Amino acids- aromatic (3)
Protein structure
B1 Amino acids- aromatic (3)
Accounts for most of UV absorbance of proteins at 280 nm hydrophobic （疏水的)
Phenylalanine (Phe, F，苯丙氨酸)
Tyrosine (Tyr, Y，酪氨酸)
Tryptophan (Trp, W，色氨酸)

11. B2 Protein structure and function
Molecular Biology Course
B2 Protein structure and function
Structure: size and shapes, primarysecondary  tertiary  quaternary, prosthetic groups (辅基，nonprotein molecules of conjugated proteins (结合蛋白)
Domains, motif, and family
Protein function

12. B2 Protein structure －Sizes
A few thousands Daltons (x 103) to more than 5 million Daltons (x 106)
Some proteins contain bound nonprotein materials (prosthetic groups or other macromolecules), which accounts for the increased sizes and functionalities of the protein complexs.

13. Complementary fit of a substrate molecule to the catalytic site
Protein structure
B2 Protein structure －Shapes
Globular proteins: enzymes
Complementary fit of a substrate molecule to the catalytic site
on an enzyme molecule.

14. Protein structure
Fibrous proteins: important structural proteins (silk fibroin, keratin in hair and wools )
Protofibril (初原纤维)
microfibril (微管)
Keratin (角蛋白)
keratin in hair

15. Formation of a peptide bond (shaded in gray) in a dipeptide.
Protein structure
B2 Protein structure －Primary
Polypeptides contain N- and C- termini and are directional, usually ranging from aa
Formation of a peptide bond (shaded in gray) in a dipeptide.

16. Protein structure
N terminus
C terminus
Structure of the pentapeptide Ser-Gly-Tyr-Ala-Leu.

17. B2 Protein structure －Secondary
a-helix
right-handed
3.6 aa per turn
hydrogen bond
N-H···O=C
A stereo, space-filling representation
Collagen triple helix:
three polypeptide intertwined

18. Protein structure
b-sheet: hydrogen bonding of the pepetide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chain
Parallel b sheet: sections run in the same direction
Antiparallel b sheet: sections run in the opposite direction
A stereo, space-filling representation of the six-stranded antiparallel b sheet.

19. B2 Protein structure －Tertiary
The different sections of a-helix, b-sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions

20. Covalent interaction: disulfide bonds
Protein structure
Noncovalent interaction between side chains that hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactions
Covalent interaction: disulfide bonds
Denaturation of protein by disruption of its 2o and 3o structure by heat and extremes of pH will lead to a random coil conformation

21. B2 Protein structure －Quaternary
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.
The quaternary structure of hemoglobin。 a1-yellow; b1-light blue; a2-green; b2-dark blue; heme-red
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22. Advantages of the quaternary structure:
Protein structure
Advantages of the quaternary structure:
It allows very large protein molecules to be made, such as tubulin。
It can provide greater functionality to a protein by combining different activities into a single entity.
The interactions between the subunits can often be modified by binding of small molecules and lead to the allosteric effects seen in enzyme regulation

23. B2 Protein structure － Prosthetic groups
Covalently or noncovalently attached to many conjugated proteins, and give the proteins chemical functionality. Many are co-factors in enzyme reactions.
Examples : heme groups in hemogobin (Figure)

24. B2 Protein structure － Domains, motifs and families
Domains: structurally independent units (tertiary) of a protein, being connected by sections with limited higher order structure within the same polypeptide. (Figure)
They can also have specific function such as substrate binding

25. Protein structure
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
bab motif

26. The primary structures of c-type cytochromes from different organisms
Protein structure
Protein families: structurally and functionally related proteins from different sources
Motif
The primary structures of c-type cytochromes from different organisms

27. The tertiary structures of the above c-type cytochromes
Protein structure
Domain
The tertiary structures of the above c-type cytochromes
Cys, Met and His side chains covalently linked to the heme to the protein
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28. Protein structure
B2 Protein functions
Enzymes: substrate binding, side chain in catalysis
Signaling: cell membrne
Transport and storage: hemoglobin transports oxygen
Structure and movement: collagen, keratin, tubulin in cytoskeleton, actin and myosin for muscle contraction
Nutrition: casein (酪蛋白) and ovalbumin(卵清蛋白)
Immunity: antibodies
Regulation: transcription factors

29. Protein structure
B3 Protein analysis
1. Purification: to obtain enough pure sample for study
2. Sequencing: determine the primary structure of a pure protein sample
3. Mass determination: determine the molecular weight (MW) of an interested protein.
4. X-ray crystallography and NMR: determine the tertiary structure of a given sample.

30. Protein structure
Protein purification
To purify the interested protein from other proteins and nonprotein molecules existing in the cells
An essential experimental step prior to study of any individual protein

31. The principal properties of proteins used for purification
Protein structure
The principal properties of proteins used for purification
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

32. Gel filtration chromatography
Protein structure
Gel filtration chromatography

33. Protein structure
Bead: the matrix determine the size of the pore

34. Protein structure
Ultracentrifugation: can be used to separate proteins according to their size and shape that determine their sedimentation rate. Very large protein complex.

35. Protein structure
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 < pI
pH=pI
pH > pI

36. Charge 1: Ion-exchange chromatography
Protein structure
Charge 1: Ion-exchange chromatography
Sample mixture + + +
Protein
binding
Ion
displacing
Column + anions
Column + anions
Column + proteins
Purified protein

37. Charge 2: Electrophoresis
Protein structure
Charge 2: Electrophoresis
Protein migrate at different position depending on their net charge +

38. Charge 3: Isoelectric focusing
Protein structure
Charge 3: Isoelectric focusing
A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel.

39. Protein structure
3. Hydrophobicity:
hydrophobic interaction chromatography
Similar to ion-exchange chromatography except that column material contains aromatic or aliphatic alkyl groups

40. 4. Affinity chromatography
Protein structure
4. Affinity chromatography
Enzyme-substrate binding
Receptor-ligand binding
Antibody-antigen binding

41. 5. Recombinant techniques:
Protein structure
5. Recombinant techniques:
Clone the interested protein encoding gene 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.

42. Mass Determination Gel filtration chromatography and SDS-PAGE
Protein structure
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.

43. Mass spectrometry:
Molecules are vaporized and ionized, and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured
extremely accurate, but expensive
MALDI can measure the mass of proteins smaller than 100 KDa
Helpful to detect post-translational modification
Protein sequencing: relying on the protein data base

44. Protein sequencing : Determine the primary structure of a protein
Protein structure
Protein sequencing : Determine the primary structure of a protein
Specific enzyme/chemical cleavage:
Trypsin cleaves after lusine(K) or arginine (R)
V8 proteease cleaves after glutamic acid (E)
Cyanogen bromide cleaves after methionine (M)
Edaman degradation:
Performed in an automated protein sequencer
Determine the sequence of a polypeptide from N-terminal amino acid one by one.
Expensive and laborious

45. Most protein sequences are deduced from the DNA/cDNA sequence
Protein structure
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

46. X-ray crystallography and NMR
Protein structure
X-ray crystallography and NMR
Determing the tertiary structure (3-D) 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 tertiary structure
Many proteins have been crystallized and analyzed

47. NMR
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

48. Protein structure Protein Folding: back
chaperones are involved in vivo
The rapidly reversible formation of local secondary structure
Formation of domains through the cooperative aggregation of folding nulcei
Assembly of domains into a “molten” globule
Conformational adjustment of the monomer
Final conformational adjustment of the dimeric protein to form the native structure.
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