Download carbonyl carbons

Biochemistry 330
September 13, 2011
BioChem 330 - Course Outline
• 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)
• Chemistry of amino acids building blocks
– Chirality
– Structure
– Functionality
– Acid/base behavior
– Polarity
• The Peptide Bond
How do we measure hydrophobicity?
• Polarity Scale ranges from +1.18 (K) to -1.68 (I)
– suggests which amino acids R groups are likely to be
exposed to solvent (pos value) and which are likely to be
buried in interior of protein (neg value)
– Based on calculations of the energy necessary to transfer
the side group from water to a less polar solvent.
– Guy, Biophysical J. 1984, 47, 61-70
• Hydropathy Scale ranges from -4.5(R) to +4.5 (I)
– Essentially the opposite of polarity scale, pos numbers
likely buried and neg numbers likely to be exposed.
– Based on analysis of where side groups tend to be found
– Kyte and Doolittle, J. Mol. Bio, 1982 157, 105-132
Thermodynamic properties of
amino acids
Name
Polarity
Hydropathy
pKa-COOH
pKa-NH2
pKa-R
Aspartate
Asp - D
+0.80
-3.5
2.09
9.82
3.86
Glutamate
Glu- E
+0.77
-3.5
2.19
9.67
4.25
Histidine
His – H
+1.18
-3.9
2.18
8.95
10.5
Lysine
Lys – K
-0.49
-3.2
1.82
9.17
6.00
Arginine
Arg - R
+0.84
-4.5
2.17
9.04
12.5
Thermodynamic properties (cont’d)
Name
Polarity
Hydropathy
pKa-COOH
pKa-NH2
Alanine
Ala - A
+0.06
+1.8
2.34
9.69
Valine
Val- V
-1.09
+4.2
2.32
9.62
Leucine
Leu – L
-1.21
+3.8
2.36
9.60
Isoleucine
Ile – I
-1.31
+4.5
2.36
9.68
Proline
Pro - P
+0.70
- 1.6
1.99
10.96
Phenylalanine
Phe - F
-1.68
+2.8
1.83
9.13
Tryptophan
Trp - W
-0.88
-0.90
2.38
9.39
Methionine
Met - M
-1.23
+1.9
2.38
9.21
pKa-R
Thermodynamic properties (cont’d)
Name
Polarity
Hydropathy
pKa-COOH
pKa-NH2
pKa-R
Glyine
Gly - G
+0.41
-0.40
2.34
9.60
Serine
Ser- S
+0.50
-0.80
2.21
9.15
13.6
Threonine
Thr - T
+.27
-0.70
2.63
10.4
13.6
Cysteine
Cys - C
-1.36
+2.5
1.71
10.8
8.3
Tyrosine
Tyr - Y
-0.33
- 1.3
2.20
9.11
10.5
Asparagine
Asn - N
+0.48
-3.5
2.02
8.80
Glutamine
Gln - Q
+0.73
-3.5
2.17
9.13
Amino acids can be post-translationally
modified
• Amino or carboxyl
group can be modified
• sugars, fats, methyl,
phosphate, sulfate
groups can be added to
side chain
• Modifications change
the way the amino acid
behaves
The polymer
Amino Acids are joined together using
Peptide Bonds to make Proteins
• Peptide bonds link amino
acids together through -NH2
on one amino acid and the –
COOH on another.
• in vivo translation is process
in which mRNA is “read” by a
ribosome, and amino acids,
each attached to a tRNA
bring the aa’s one at a time
to the active site.
• In vitro, protein synthesis can
be done by a sequential
process done on a machine.
Peptide Bond is Planar
* The peptide plane consists of six atoms, Ca1, C, O, N, H, Ca2
* H-N-Ca bond angle is 121o, not 109.5o.
Resonance in Peptide Bond
..
The peptide bond is rigid and planar due to the double
bond resonance form, which shortens the C-N distance to
0.16 A shorter than a normal C-N bond.
Properties of Peptide Bond
• Barrier to Rotation about C-N bond is ~ 20 kcal
• Peptide bond is planar with Ca groups mostly trans
to the peptide bond (less steric clash of -R groups)
• Ca groups are tetrahedral (3-D) and able to rotate
though some angles are unfavorable due to steric
repulsion with other atoms.
• Ca are chiral are ALMOST exclusively the L
stereoisomer.
Peptides Conventional Nomenclature
• N (amino) terminal on the left
• C (carboxy) terminal on the right
• biological synthesis of proteins on the ribosome
occurs from N to C terminus.
• Amino terminal amino acid is named residue 1.
What are the thermodynamics
of peptide bond formation?
• G+A
= G-A + H2O
DGo = -RTlnK
DGo = +5 kcal/mole
DGo = -RT ln [Prod]/[Reac]
– [Prod]/[React] = exp(-5000/RT) ≈ exp(-8) ≈ 3.3 x 10-4
– [Prod]/[React]= dipeptide/free amino acids ≈ 1/3000
• Only 1 in 3000 dipeptide bonds should remain intact
at equilibrium in solution
• Remaining peptide bonds should hydrolyze and
exist as free amino acids!
• Why don’t they?
But first, why is hydrolysis favored
thermodynamically?
• Peptide Bond enthalpy doesn’t compensate for the
increase in entropy gained by making two
molecules rather than one molecule
• Individual amino acids may also be better solvated.
If hydrolysis is favored, why don't peptide
bonds in the proteins in our body
spontaneously undergo hydrolysis?
Activation Barrier to Hydrolysis must be greater than kT
Not only are peptide bonds created
constantly in our bodies, the average
lifetime of peptide bond is 100 years!
• How are they made?
– Thermodynamic strategy
• Peptide bond formation is coupled to the breakdown
of a high energy phosphate bond in a protein factory
known as the ribosome.
• How are they maintained?
– Kinetic strategy
• Enzymes which lower the activation barrier between
product and reactant. For the hydrolysis of peptide
bonds, these enzymes are called digestive
enzymes.
Peptide Backbone
• three atoms from each amino acid on backbone
– nitrogen,
– alpha carbon,
– carbonyl carbon.
• other atoms of the polymer hang off of this chain.
• This polymer has certain structural features which
are invariant
Summary of invariant backbone
properties
•
•
•
•
All amino acids have an L chirality at Ca
Peptide bond itself is planar
No rotation about peptide bond
Ca on adjacent amino acids are most often
trans to the peptide bond.
– proline can be cis or trans
Where does flexibility occur?
• Variation in the 3-D structure of a protein is a result of
rotational freedom about the sp3 hybridized Ca
• Rotations of the backbone result in very different
polymers and involve the relative orientation of two
contiguous peptide planes
What is a dihedral angle?
• Dihedral angle is the angle
– between four atoms
• (Ha, C1, C2, Hd)
– or across three bonds
• (Ha-C1- C2- Hd)
• It is measured by sighting
down the central bond, or
the central two atoms, and
looking at the through space
angle made by the atoms of
the first bond relative to the
atoms of the third bond.
C1
C2 is eclipsed
Dihedral angle in Ethane
H3C-CH3 here is 60o
Backbone Dihedral Angles
f, Phi rotation:
– the dihedral angle phi, f, represents the rotation about N-Ca
– The four atoms which define f are the carbonyl carbonn-1,
the nitrogenn, the a-carbonn, and the carbonyl carbonn.
– Zero for f is where the two carbonyl carbons come into
closest contact with one another.
F
Backbone Dihedral Angles
y Psi rotation:
– the dihedral angle psi, y., measures rotation about Ca-C
– The four atoms which define y are the nitrogenn, the acarbonn, the carbonyl carbonn and the nitrogenn+1.
– Zero for y is where the two nitrogens come into closest
contact with one another.
y
Backbone Dihedral Angles
• Rotations of f and y by definition are from the
perspective of the chain direction (N to C terminal)
– rotations are (+) clockwise; (-) counterclockwise
• N-Ca for f
• Ca-C for y
• Stupid Pneumonic
• Phi includes the n-1 residue, with the n amino acid saying
to the n-1 amino acid, you're [p]history.
• Psi includes the n+1 residue, with the n carbonyl
[p]sighting ahead to the n+1 amino acid.
Other degrees of Freedom
• Rotations of the Side Groups
– -R groups of the amino acids show a great deal of
variability in non-backbone dihedrals
– prolines
• can only adopt certain phi,psi angles and so are not found
in some types of secondary structure.
• It is nearly impossible to twist a proline into an alpha
helical orientation, thus it is known as a helix breaker.
Molecular Architecture Secondary Structure
• Ramachandran Plots are contour plots of the
conformational energies as a function of psi (x-axis)
and phi (y-axis) values for alpha carbons in protein
backbones. RAMACHANDRAN GN, RAMAKRISHNAN C, SASISEKHARAN V., J Mol Biol., 7:95-99
•
http://dicsoft1.physics.iisc.ernet.in/rp/
Beta Sheet
Alpha helix
Ramachandran plot
Blue: helix
Red: strand
Green: turn and loop.
Topological Lines
outer, 90% probab.
Inner, 50% probab.
+ non-gly aa
X gly
□
prolines.
PDB NOSynthase
Protein Structure Molecular Architecture
• Phi and Psi Dihedrals
4
n+1
3,4
2,3
1,2
1
n-1
n
– Phi rotation, f : the rotation of the
trailing polypeptide about the Ca -N
bond. The four atoms which define f
are (1) the carbonyl carbonn-1, (2) the
nitrogenn, (3) the a-carbonn, and (4)
the carbonyl carbonn.
– Psi rotation, y: the rotation of the
leading polypeptide about the C"-C
(carbonyl) bond. The four atoms
which define y (1) the nitrogenn, (2)
the alpha carbonn, (3) the carbonyl
carbonn, and (4) the nitrogenn+1.
Secondary Structure a-HELIX
• a-HELIX, predicted by Pauling in 1948 and
found experimentally by Kendrew in the crystal
structure of myoglobin in 1953.
– 3.613 right handed a-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 f -60o, y -50o
Protein Structure a-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.
Protein Structure a-HELIX
13 atoms between hydrogen bonds.
Protein Structure
α-HELIX
* 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 myoglobin file with Rasmol
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 Cterminus,
• leads to a depression in the pKa of K or R at the Nterminus.
Protein Structure α-HELIX
Dipole Moments All Line Up
Protein Structure α-HELIX
Protein Structure α-HELIX
•
Physical Length:
– Typical length of alpha αhelix is about 10 residues or
15 Å, remember 1.5 D
/residue) though there's quite
a range. Though it used to be
thought that helices in
proteins rarely got larger than
100 D
– Now, images of proteins like
tropomyosin at right reveal
much larger helices that are
helices of helices.
See Tropomyosin ent files
using Rasmol
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-SCH2-.
– A, E, L and M are good alpha
helix formers
R groups on the outside of helix:
– P, G, Y and S are poor helix
formers
Protein Structure α-HELIX
• Human glucocorticoid receptor
– pdbID 1M2Z
glucocortisone
dexamethasone