biochem 330 - course outline

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BioChem 330 - Course Outline September 15, 2011

•  Bio-molecular Structure/Function (I) –  PROTEINS

•  Structure – Chemistry of amino acid building blocks –  Primary, secondary and tertiary structure –  Protein folding, thermodynamics and kinetics –  Predictions of protein folding, dynamics

•  Function –  Binding ….a tale of two globins (hemoglobin and

immunoglobulin)

Protein Structure Molecular Architecture •  Phi and Psi Dihedrals

–  Phi rotation, φ : the rotation of the trailing polypeptide about the Cα -N bond. The four atoms which define φ are (1) the carbonyl carbonn-1, (2) the nitrogenn, (3) the α-carbonn, and (4) the carbonyl carbonn.

–  Psi rotation, ψ: the rotation of the leading polypeptide about the C"-C (carbonyl) bond. The four atoms which define ψ (1) the nitrogenn, (2) the alpha carbonn, (3) the carbonyl carbonn, and (4) the nitrogenn+1.

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3,4

1,2

2,3

1

n-1

n

n+1

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Secondary Structure α-HELIX

•  α-HELIX, predicted by Pauling in 1948 and found experimentally by Kendrew in the crystal structure of myoglobin in 1953. –  3.613 right handed α-helix, which means that there are

3.6 residues per turn, and 13 atoms between hydrogen bonds.

–  5.4 repeat unit, the length of one turn in an alpha helix. –  5.4/3.6 = 1.5 Å/residue. –  dihedrals of approximately φ -60o, ψ -50o

Protein Structure α-HELIX •  Favorable H-Bonding exists between

the O of one amino acid residue and the N-H of a different amino acid residue four amino acids away. –  A H bond is created when the electronegative lone

pair electrons on the carbonyl oxygen cause the amide H to shift slightly away from N and towards O and form a weak bond.

–  O- - - H-N bond distance is ~2.9 Å –  bond energy is ~ 10 kJ/mole or less (compared

with 400kJ for a C-H bond).

•  Must remember that these atoms would be interacting with solvent if they weren’t involved in this internal H bonding.

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Protein Structure α-HELIX

13 atoms between hydrogen bonds.


Myoglobin Structure in Jmol and also in Proteopedia

* In this highly idealized image of myoglobin, all of the helices are represented as colored ribbons, which trace their backbone.

* For simplicity, all side groups are completely ignored.

• How many helices do you see?

• See pdbID 1mbc and 1a6m

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Protein Structure α-HELIX •  Large Dipole Moment Along Helix Axis

–  is created by the alignment of the dipoles of each peptide along the helix axis.

–  is positive at the amino end and negative at the carboxy end of the helix.

–  3.5 DeByes/peptide bond; 0.7 kcal/mole •  Ref: Wada, Adv. Biophysics 9 1-63 (1976)

–  D or E stabilize helix when at N-terminus but destabilize helix what at C-terminus,

•  leads to depression in the pKa of D or E at N-term.

–  K or R destabilize at N-terminus, stabilize at C-terminus,

•  leads to a depression in the pKa of K or R at the N- terminus.


Dipole Moments All Line Up

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Protein Structure α-HELIX •  Physical Length:

–  Typical length of alpha α-helix is about 10 residues or 15 Å, remember 1.5 Å/residue) though there's quite a range. Though it used to be thought that helices in proteins rarely got larger than 100 Å

–  Now, images of proteins like tropomyosin at right reveal much larger helices that are helices of helices.

See Tropomyosin (pdb 1C1G) files using rcsb in PDB or in proteopedia.

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Protein Structure α-HELIX •  -R groups big and bulky

side groups accommodated –  charged amino acids project

away from the cylinder –  CH2SH group of a C can

combine with a -CH2SH side group of a second C to make a disulfide bond, -CH2S-S-CH2-.

–  A, E, L and M are good alpha helix formers

–  P, G, Y and S are poor helix formers

R groups on the outside of helix:

Beta Structures

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β strands form the basic unit from which spider silk is made

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Protein Structure β Strand •  Characteristics of β

strands –  When Φ,Ψ : approximately -140, +135 for the antiparallel

beta sheet –  3.4 Ǻ per residue means that

this is a fully extended structure

–  -R groups stick alternatively up above the peptide plane and down below the peptide plane.

Exploring β strands w Jmol •  What is the name of this polymer? •  Are the dihedral angles for the end

residues the same as for the internal residues?

•  What is the end to end distance of this polymer and how does it compare with the a helix?

•  Are all the peptide bonds “trans” in this structure?

•  What would the amino protons and carbonyl oxygens be H bonding with if this were by itself in solution?

•  What would the amino protons and carbonyl oxygens be H bonding with if this were in a protein?

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How do the strands assemble into a sheet? •  1. Anti parallel Beta Sheet:

–  If the strands run in opposite N→C directions, then the sheet is anti parallel.

–  H bonds are made between different strands of the sheet, inter-strand H bonded network.

–  The H bonding pattern in this sheet is straight across with a narrowly spaced pair followed by a wide space. (can vary)

–  R groups: •  alternate up and down the strand and

across each strand, •  are both up or both down across

strands.

Protein Structure Beta Sheet •  Beta sheet predicted (once again) by Pauling and

found later experimentally in proteins. –  Typical protein with extended beta sheet structure is

fibroin, the major protein in silk which is a fabric produced by the silkworms of China and used for fabric since approximately 2,000BC.

Here, silk worms munch on mulberry leaves. You can find coordinates for silk under Blackboard silk.ent coordinates.

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Protein Structure* Silk Note the H bonding within sheet that is inter-strand, and spacing pattern.

Strand 1a

Strand 2a

Sheet A

Strand 1b

Strand 2b

Sheet B

Protein Structure* Silk Note the R groups project above and below the sheets, and that the sheets aren’t perfectly flat, but are puckered in such a way that the knob of one R group fits into the upward pucker of sheet above.

Intersheet van der Waals interactions govern polymer properties

Identity of R groups determine tensile strengths, flexibilities, and price of different silks.

Sheet A

Sheet B

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Sequence Determines Properties •  Molanari Silk worms produce fibers with a. a.

sequence alternating ( Gly-Ala-Gly-Ala-Gly-Ser) -etc. –  With this sequence, all the Gly -R groups are on

one side of the sheet, and all of the Ala or Ser R groups are on the other side of the sheet.

–  Two sheets pile on top of each G/G and A/A resulting in a very flexible, soft fabric, as the only thing holding one sheet to another is the weak van der Waals attractive forces between the R groups.

–  Meanwhile within the plane of the sheet, strong covalent and hydrogen bonding forces result in a strong, inextensible fabric.

Exploring the Structure of Silk •  Are the H bonds…...

–  Spaced regularly along the strand?

–  Directed to the same strand in the same peptide bond?

•  What is the distance between strands? …..between sheets?….end to end?

•  Is the structure modeled in silk antiparallel?

•  How are the R groups arranged…. –  With respect to the strand? –  Across the sheet? –  intersheet?

B sheet structure of silk

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Sequence Determines Properties

From the world of polymer science, we see the same principle emerging. Here, look at the structure of the miraculously strong polymer, Kevlar

Protein Structure Beta Sheet •  Adjacent strands run in

the same direction (-N to -C terminal), then the sheet is parallel. –  H bonding pattern is

evenly spaced but the individual bonds are angled across the two strands.

–  Values Φ,Ψ : approximately

–  -119, +113 for the parallel sheet

Parallel Beta Sheet:

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Protein Structure Molecular Architecture

•  Beta twists: a common structural motif in which a mostly parallel beta sheet naturally twists.

Protein Thioredoxin

The twisted sheet is not purely parallel or antiparallel , but has a mixed orientation.

Note from the N-term.: strand/loop/helix/loop strand/loop/helix/loop repeat


•  Beta twists II

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•  Beta twists III


Twisted beta sheets are found as the central structural feature of many, many diverse proteins.

This beta stranded scaffold is then derivatized with loops and helices which are the key to function.


•  Beta Barrels GFP, green fluorescent protein This beta stranded barrel and tight loops create a shielded lantern to hold the fluorescing group (cyclized triad of amino acids shown below). If group were exposed to solvent, the light would be extinguished.

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Protein Structure Loops •  Loops: Making the Connections

–  Loop regions connect regions of secondary structure, either helices or strands, or helices to strands. Previously thought to be unstructured random arrangements of amino acids, it is now realized that some regular features exist which can be described and in some cases rationalized.

–  They are usually at the surface of the molecule and therefore have polar charged or hydrophilic polar side groups. G is another favorite. Surprisingly, one can predict loop regions with more certainty than any other "structure". The carbonyl oxygen and amide proton hydrogen bond with the solvent.

Protein Structure Loops •  Making Connections

between α Helices –  Alpha helices are often

connected to each other by loop regions as seen in Mb.

–  The carbonyl and amide groups in the loops make hydrogen bonds with the solvent rather than internally.

–  Loop regions are often the sites of mutations, insertions, and deletions, since it seems you can perturb the loop and not affect the core structure. Myoglobin again, see loops

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Protein Structure Loops •  Making the Connections •  Functional Loops

–  Often find active sites or binding sites at loop regions, accessible to solvent, and containing many polar charged groups which usually serve to bind substrate or metals. An example is the helix-loop-helix of the calcium binding proteins. See calmodulin 1cll in pdb

or in Proteopedia

Protein Structure Loops •  Making Connections

between β strands –  Antigen binding site in an

immunoglobulin is built up from 6 loop regions attached to an invariant, genetically conserved beta structure core.

–  Again, these are sites for mutations to turn antibody specificity from one antigen to another

See antibody file 1IGT

In pdb or proteopedia

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Protein Structure Loops •  LOOPS CONNECTING TWO ANTI BETA SHEETS:

–  are usually fairly short since the two strands they connect are fairly close together.

–  Common arrangement is the hairpin loop, which uses two amino acids to turn the strand completely. Two sets of phi,psi describe Type I and Type II turns.

•  the peptide bond between the two connecting amino acids is perpendicular to the strand direction, and hydrogen bonding occurs with the solvent. In one case, the oxygen projects above the plane, nitrogen proton below and in the other, the reverse is true.

•  Also true is that the register of the disposition of the side group usually remains intact, that is, residues across from each other both project above the plane or below the plane of the strand.

Protein Structure Loops

LOOPS CONNECTING TWO ANTIPARALLEL BETA STRANDS:

biochem 330 - course outline

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