1. Lecture 11
Test next week in class
Protein structure

2. Collagen Most abundant protein of vertebrates.
Extracellular protein-insoluble fibers, great tensile strength
Major component of connective tissues (bone, teeth, cartilage, tendon, ligament, etc.)
Type I collagen-3 polypeptide chains, 285 kD.
3000 Å long and 14 Å diameter.
Distinct amino acid composition; 1/3 are Gly and 15-30% are Pro and 4-hydroxyprolyl (Hyp) residues.

3. Collagen Collagen has a triple-helical structure
Amino acid sequence has repeating triplets of Gly-X-Y with X=Pro and Y= Hyp over 1011 residues out of 1042 residue polypeptide.
Forms a right-handed triple helical structure.

4. The triple helix of collagen.
Shows how left-handed polypeptide helices are twisted together to form a right-handed superhelical structure.
Individual polypeptide has 3.3 residues per turn and pitch of 10 Å.
The collagen triple helix has 10 Gly-X-Y units per turn and a pitch of 86.1 Å.
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5. Figure 8-30b X-Ray structure of the triple helical collagen model peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (b) View along helix axis.
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6. Figure 8-31 Electron micrograph of collagen fibrils from skin.
Collagen is organized into distinctive banded fibrils. These fibrils have a periodicity of 680 A and a diameter of 100 to 1000 Ang depending on the type of collagen they contain.
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7. Figure 8-32 Banded appearance of collagen fibrils.
The banded appearance in an elctron micrograph arises from the staggered arrangement of collagen molecules. This space is about 680 angstrom s so that a 3000 angstrom long molecule is about 4.4D.
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8. Figure 8-30c X-Ray structure of the triple helical collagen model peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (c) A schematic diagram.
The well packed rigid triple helical structure is responsible for the charcteristic tensile strength of collagen.
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9. Collagen fibrils are covalently cross-linked.
Figure 8-33 A biosynthetic pathway for cross-linking Lys, Hyl, and His side chains in collagen.
Collagen fibrils are covalently cross-linked.
Collagen almost no cysteine.
It is cross linked y Lys and His.
Lysyl oxidase coverts lysine to allysine.
Allysine are condesed to allysine aldol.
This reacts with His to form Aldol-His.
Aldol-His reacts with 5-hydroxy-Lys crosss linking the four side chains.
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10. Table 8-3 The Arrangement of Collagen Fibrils in Various Tissues.
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11. Globular proteins
Diverse group of proteins that exist as compact spherical molecules.
Enzymes, transport, and receptor proteins.
Most structural information from X-ray crystal structure and NMR.
X-ray crystallography directly images molecules.
X-ray wavelengths are small 1.5 Å (visible light is 4000 Å)
X-rays generated by synchrotrons, a type of particle accelerator to make X-rays of high intensity.

12. Crystalline proteins
Molecules in protein crystals are arranged in regularly repeating 3-D lattices.
Unlike other small organic or inorganic molecules, proteins are highly hydrated (40-60% H2O)
Water is required for the native structure of the proteins.
Generally disordered by >1 Å.
Typical resolution is 1.5 to 3.0 Å.

13. Figure 8-36a Electron density maps of proteins.
Electon density maps are presented as a series of parallel sections through the object. Each section the electron density is represented by contours in the same way contours on a topographic map.
Section of 2.0 A resolution elecron density map with the heme group.
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14. Figure 8-36b Electron density maps of proteins.
Portion of 2.4A resolution electron density map made with contoured transparencies.
Dots are nonhydrogen groups.
The heme group is seen edge on together with the two His residues that are associated withj it.
An alpha helix (E helix is shown along the bottom). Another alpha he3lix (C-helix is seen extending into the plane in the upper right hand corner.
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15. Figure 8-36c Electron density maps of proteins.
A thin sectio through the 1.5A resolution electron denisty map of E. coli 6-hydroxymethyl-7,8 dihydropterin pyrophosphokinase (catalyzes the first step in the production of folic acid).
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16. Figure 8-37 Sections through the electron density map of diketopiperazine calculated at the indicated resolution levels.
Electron density map is interpreted from atomic positions, accuracy depends on the crystal resolution limit.
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17. Crystalline proteins
Crystalline proteins assume the same structure they have in solution
Crystals have 40-60% water content (similar to most cells)
Proteins may crystallize in of several forms depending on conditions. Different crystal forms of the same protein have identical conformations.
Many enzymes are catalytically active in the crystalline state.

18. NMR for protein structure determination
Use of 2D NMR
Yields interaatomic distances between specific protons that are <5 Å apart.
Interproton distances through space can be determined by nuclear Overhauser effect spectroscopy (NOESY)
Interproton distance through bonds as determined by correlated spectroscopy (COSY).
Present methods are good only with molecular masses up to 40 kD.
Usually well correlated with X-ray data, but sometimes differs.
NMR can probe motions over time scales of 10 orders of magnitude so can be used to study protein folding and dynamics.

19. Nuclear Overhauser Effect (NOE)
Figure 8-38a The 2D proton NMR structures of proteins. (a) A NOESY spectrum of a protein presented as a contour plot with two frequency axes w1 and w2.
Off diagonal peaks (cross peaks) occur from interaction of 2 protons that are <5 Å apart in space and whose 1D-NMR peaks are located where the horizontal and vertical lines cross through the cross peak intersect the diagonal.
Nuclear Overhauser Effect (NOE)
Conventional 1D-NMR spectrum of the protein occurs along the diaonal of the plot where omega 1 = omega 2 is too crowded with peaks to e interepreted.
The off diagonal peaks (cross peaks) arise from interaction of 2 protons that are <5A apart in space and whose 1D NMR peaks are located where the horizontal and vertical lines through the cross peak interesect the diagnol. (NOE).
The line to the left of the spectrum represents the extended polypeptide chain with its N and C terminal ends identified by N and C and the positions of 4 protons a-d are represented by small circles. The dashed arrows indicate the diagonal NMR peaks wich these protons give rise.
Cross peaks such as I, j, and k which are each located at the intersections of the horizontal and vertical lines through 2 diagonal peaks are indicative of an NOE between the 2 protons (meaning they are less than 5A apart.
These distance relationships are indicated by the three looped structures below the spectrum. The assignment of distance relationship between two protons of a given polypeptide requires that the NMR peaks to which thhey give rise and their positions in the polypptide be known. c b
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20. Figure 8-38b. The 2D proton NMR structures of proteins
Figure 8-38b The 2D proton NMR structures of proteins. (b) The NMR structure of a 64-residue polypeptide comprising the Src protein SH3 domain.
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21. Tertiary structure
Tertiary structure is the three dimensional arrangement of a protein.
Includes the folding of secondary structural elements and spatial dispositions of the side chains.
Determined by X-ray crystallography and NMR

22. Figure 8-39a Representations of the X-ray structure of sperm whale myoglobin. (a) The protein and its bound heme are drawn in stick form.
The first X-ray structure was that of myoglobin. This is the ball and stick form. You can see the heme group in the cnter with the Fe as the sphere.
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23. Figure 8-39b Representations of the X-ray structure of sperm whale myoglobin. (b) A diagram in which the protein is represented by its computer-generated Ca backbone.
A diagram with a computer generated alpha carbon backbone. The carbon atoms are shown as balls here. The proteins bound heme group is shown.Part of the heme has been displaced for clarity here.
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24. Figure 8-39c Representations of the X-ray structure of sperm whale myoglobin. (c) A computer-generated cartoon drawing in an orientation similar to that of Part b.
A computer generated cartoon drawing in ain an orientation similar to the previous drawing. The heme group with its bound oxygen molecule and the His side chains are shown as ball and stick here.
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25. Globular proteins have both  helices and  sheets
Most proteins have a significant amount of secondary structure
On average 31%  helix, 28%  sheet, and a total content of helices, sheets, turns and  loops comprising 90% of the structure of a protein.

26. Figure 8-40 The X-ray structure of jack bean protein concanavalin A.
This protein is largely antiparallel b pleated sheet (represented by the flat arrows which point towards the proteins C terminus. The balls are metal ions.
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27. Figure 8-41 Human carbonic anhydrase.
Alpha helices are represented as cylinders and each strand of b-sheet is drwan as an arrow going towards the C-terminus. The gray ball is a Zn ion with three His side chains around it.
Note that the C terminus is tucked through the plane of a surrounding loop so that carbonic anhydrase is one of the rare native proeins in which a polypeptide forms a knot.
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28. Side chain location varies with polarity
Globular proteins lack the repeating sequences responsiblee for the regular conformations of fibrous proteins.
The amino acid side chains in globular proteins are distributed according to polarities.
Nonpolar residues (Val, Leu, Ile, Met, and Phe) occur in the interior of a protein.
Charged polar residues (Arg, Lys, His, Asp, Glu) are mostly located on the surface of a protein.
Uncharged polar residues (Ser, Thr, Asn, Gln, Tyr, and Trp) are usually on the surface but can occur in the interior of the protein.
If in the interior, they are H-bonded to neutralize their polarity.

29. Figure 8-42a The X-Ray structure of horse heart cytochrome.
Hydrophobic side chains are shown in red. In b the hydrophillic side chains are shown in green.
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30. Figure 8-43a. The H helix of sperm whale myoglobin
Figure 8-43a The H helix of sperm whale myoglobin. (a) A helical wheel representation in which the side chain positions about the a helix are projected down the helix axis onto a plane.20
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31. Figure 8-43b. The H helix of sperm whale myoglobin
Figure 8-43b The H helix of sperm whale myoglobin. (b) A skeletal model, viewed as in Part a.
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32. Figure 8-43c. The H helix of sperm whale myoglobin
Figure 8-43c The H helix of sperm whale myoglobin. (c) A space-filling model, viewed from the bottom of the page in Parts a and b and colored as in Part b.
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33. Figure 8-44 A space-filling model of an antiparallel b sheet from concanavalin A.
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34. Energy diagram of the protein folding process.
The completely unfolded protein is thought to be in the least stable form.
For most proteins, the native conformation is the most thermodynamically stable and the only form that is biologically active.

35. Denaturation and renaturation of a protein
The complete loss of organized structure in a protein is called “denaturation”.
Denaturation results in loss of biological activity!
Denaturation process occurs during cooking an egg.
Denaturants include:
Large evil fire ants??
Heat
Organic solvents
Urea
Detergents
Acid or base
Shear stress
Hydrophobic interfaces

36. Structural Motifs in Proteins.
Individual units of 2ndary structure combine into stable, geometrical arrangements.
Called supersecondary structure or motifs.
Are often repeated in same protein, different proteins.
Certain motifs have associated biological functions:
a-Helix-loop-a-helix motif binds DNA, sequesters calcium ion.
Secondary structures often depicted as ribbon diagrams
Ribbons invented by Jane Richardson, originally drawn by hand, now done by computer programs.

37. The aa motif (helix-turn helix)
Some common structural motifs of folded proteins
The aa motif
(helix-turn helix)
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38. b) The bb motif; antiparallel
Some common structural motifs of folded proteins
b) The bb motif; antiparallel
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39. c) The bbbb “Greek Key” motif
Some common structural motifs of folded proteins
c) The bbbb “Greek Key” motif
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40. d) The bab motif Some common structural motifs of folded proteins
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41. Several bab motifs combine to form a superbarrel in the glycolysis enzyme triose phosphate isomerase (TIM barrel)
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42. Quaternary structure Spatial arrangement of protein subunits.
Polypeptide subunits associate in a geometrically specific manner.
Why subunits?
Easier to repair self-assembling single subunit vs. a large polypeptide.
Increasing a protein’s size through subunits is more efficient for specifying the active site.
Provides a structural basis for regulating activity.

43. Domains in proteins.
Common sequence regions in native proteins can fold up to form compact structures called “domains”.
Domains can range in size from amino acids, have upper limit in forming compact hydrophobic core.
Domains are a type of folding motif, typically have separate hydrophobic core.
Larger proteins are composed of multiple domains, often connected by flexible linker peptide regions.
Classic example: antibodies

44. Antibody Immunoglobulin Domains
Structural elements of IgGs:
Naturally occurring immunoglobulins (IgG molecules) have identical heavy chains and light chains giving rise to multiple binding sites with identical specificities for antigen.

45. Antibody Immunoglobulin Domains
Antibodies are composed of:
V (for variable) regions - encodes the antigen binding activity
C (for constant) regions - encodes immune response signal/effector functions:
Complement fixation (activation of complement cascade)
Binding and activation of Ig receptors (transport from maternal source, activate immune system T cells to engulf, destroy foreign cells, particles, proteins)
Also binds bacterial Protein A, Protein G (used in purification)
Note: dashed lines indicate
interchain disulfide bonds

46. Antibody Immunoglobulin Domains
There is a conserved glycosylation site in the CH2 domain of IgG (purple region).
A carbohydrate is covalently attached here by postranslational modification.

47. Antibody Immunoglobulin Domains
IgG secondary/tertiary structure: multiple beta-sheet domains.
Termed “immunoglobulin domain”.
Repeated motif in many immune and receptor proteins.

48. Antibody Immunoglobulin Domains
Modes of Flexibility of IgG structure

49. Subunit interactions
Identical subunits in a protein are called protomers
Proteins with identical subunits are oligomers.
Hemoglobin is a dimer (oligomer of two protomers) of protomers.