Transcript College 4

College 3
Macromoleculen en biomoleculen
Maar eerst:
elektron configuratie Cytosine
DNA base pairing
•A with T: adenine (A) always pairs with thymine (T)
•C with G: cytosine (C) always pairs guanine (G)
This is consistent with there not being enough space (20 Å) for two purines to fit within the helix and too much
space for two pyrimidines to get close enough to each other to form hydrogen bonds between them.
But why not A with C and G with T?
The answer: only with A & T and with C & G are there opportunities to
establish hydrogen bonds (shown here as dotted lines) between them
(two between A & T; three between C & G).
These relationships are often called the rules of Watson-Crick base
pairing, named after the two scientists who discovered their structural
basis.
The rules of base pairing tell us that if we can "read" the sequence of
nucleotides on one strand of DNA, we can immediately deduce the
complementary sequence on the other strand.
H-Bonds
The hydrogen bond
Fig. 2.2.5.A hydrogen bond between two water molecules. The
strength of the interaction is maximal when the O-H covalent
bond points directly along a lone-pair electron cloud of the
oxygen atom to which its hydrogen bonded.
In het algemeen: D—H · · · ·A
The large electronegativity difference between H and O confers a 33% ionic
character on the OH-bond as reflected by water’s dipole of 1.85 debye units.
→ highly polar molecule
The electrostatic interactions between the dipoles of two water molecules tend to
orient them such that the O-H bond on one molecule points towards a lone pair
electron cloud on the oxygen atom of the other water molecule
The hydrophobic interaction
Because these cage structures are more ordered than the surrounding water,
their formation increases the free energy. This free energy cost is minimized,
however, if the hydrophobic (or hydrophobic parts of amphipathic molecules)
cluster together so that the smallest number of water molecules is affected.
The hydrophobic interaction
in membranes
The hydrophobic interaction
in proteins
Macromolecules and Biomolecules
Levels of structure
Natural and synthetic polymers
Primary structure: sequence of small molecular residues that make up the
polymer
proteins are formed from 20 different amino acids strung together by
the peptide bond, -CONH- (see below). The determination of the primary
structure is called ‘sequencing’.
The secondary structure of a macromolecule is the (often local) spatial
arrangement of a chain
random coil, helices, sheets
The tertiary structure is the overall three-dimensional structure of a
macromolecule
The quarternary structure of a macromolecule is the manner in which large
molecules are formed by aggregation
Random coil
•
The most likely conformation of a chain of identical subunits not capable of forming
H-bonds or any other type of specific bond is a random coil. Polyethylene is a simple
example. The random coil model is a helpful starting point for estimating the orders of
magnitude of the hydrodynamic properties of polymers and denatured proteins in
solution. The simplest model of a random coil is a freely jointed chain, in which any
bond is free to make any angle with respect to the preceding one (fig 3.3)
The Structure of Proteins
Proteins are the ‘hydrogen atoms’ of life.
(Un-)Fortunately there are many of them, all of them have a specific function and
as a consequence we have to figure out the rules by generalizing their physical
(and chemical and biological) properties.
A protein is a polypeptide composed of covalently linked -amino acids,
NH2CHRCOOH, where R is one of only twenty possible groups. The resulting
sequence of R groups linked by peptide bonds for a large part determines the
structure and function of the protein.
C = koolstof
N = stikstof
O = zuurstof
H = proton
R = een aminozuur
Peptide
α-helix
Eiwit
Quarternary structure,
hemoglobine
The Structure of Proteins
R = 20 different amino acids, ‘simple’ organic molecules composed of C, H, N, O
and an occasional S.
Nevertheless, the chemical properties of the various amino acid side chains vary
from hydrophobic to polar to charged, from large to small, from flexible to rigid and
these properties are used to add ‘functionality’ and ‘activity’ to a sequence of
amino acids folded into a protein structure
The amino acids
Fig 4. The basic, acidic, uncharged and non-polar sidechains.
The uncharged polar side chains are often involved in hydrogen
bonding. The hydrophobic side chains occur in the interior of a
protein and their size and shape play an important role in the
compactness of a protein. In regions where a-helices fold over
one another small residues are required. Proline is special
because it can not fit in the a-helix. Aromatic residues often have
additional functions. For instance in photosystem 2 of
photosynthesis a tyrosine plays a crucial role in electron and
proton transfer. The S atoms in methionine and cysteine play
important roles in cofactor binding and protein folding.
The electronic structure of the peptide bond
Sequence → conformation → function
Prediction of the conformation from the primary structure, the so-called
protein folding problem, is extraordinarily difficult and is the focus of much
research
One major factor determining the secondary structure of proteins is found in the
stabilization of certain structures by hydrogen bonds involving the peptide
bond.
For peptide structures Pauling and Corey (1951) proposed (without having ‘seen’
them, based on valence, LCAO, Huckel) that:
•
•
•
•
The four atoms involved, O, C, N, H lie in a relatively rigid plane.
The planarity is due to the delocalization of π-electrons over the N, C and O atoms
and the maintenance of maximum overlap of the contributing π-orbitals.
Two types of structures exist, helices and sheets, where all NH and CO groups
are engaged in hydrogen bonding.
The N, H and O atoms involved in H-bonds between different parts of a
polypeptide chain lie in a (more or less) straight line (with displacements of H
tolerated up to not more than 30o from the N-O vector.
Fig. 4.9 The peptide bond, which is an
essential part of the amino acid chain
constituting a protein, is composed of the
atoms O, C, N, H, which all are positioned in
one plane. Also the two a-carbon atoms
flanking the peptide bond are in that plane.
Consequently the configuration of the
polypeptide backbone is described by two
angles per residue indicated in the figure.
Peptide bonds
Fig 4.10 Bond angles in the
peptide bond.
Note that all the angles are
close to 120o, typical for sp2hybridization.
O-atom:
 
1
1s 2 sp 2  sp
2
4
1s 2 2 p y2O sp   1sp
2
1
2 2 pz
, sp
O
O
2
O , spC
C
2 p1zO
1πe
C-atom:
1s 2 1sp2 sp  1sp2 sp3  1sp2 sp2 2 p1z
1πe
N-atom:
1s 2 1sp2 ,sp2  1sp2 ,sp3  1sp2 ,1s 2 p z2N
2πe
C,
N
O
C,
C
Cai
N
C,
Cai 1
N
N
H
Π orbitals..
linear combination of the 2 p zO ,2 p zC ,2 p z N orbitals, results in 3 π orbitals
Accommodating the 4 π electrons
Bonding energy from π electrons makes the peptide bond rigid and planar.
Π network does not extend over Cα
The total energy of a protein and its energy
landscape
The simplest calculations of the conformational energy of a polypeptide.
1. Bond stretching. model a bond as a spring, then the potential energy takes the
form of Hooke’s law and is given by:
2
Vstretch  1 k stretch R  Re 
(4.16)
2
2. Bond bending. An O-C-H bond angle may open out or close in slightly
to enable the molecule as a whole to better fit together. If the equilibrium bond angle is we
2
write:
Vbend  1 k bend    e 
2
(4.17)
where is the bending force constant, a measure for how difficult it is to change the bond
angle. Again this contribution must be summed
allcos
bonds.
Vtorsion over
 A1 
3   B1  cos 3 
3. Bond torsion.
(4.18)
Because for a regular structure, like an -helix only two angles are needed to specify the
conformation of that helix, and they range from -180o to +180o, the torsional potential energy
of the entire molecule can be represented on a Ramachandran plot, a contour diagram in
which one axis represents and the other represents .
For a right-handed α-helix
  57 o and   47 o.
Plus
4. Interaction between partial charges.
5. Dispersive and repulsive interactions, Lennard-Jones potential.
6. Hydrogen bonding.
α-helix
Fig.4.13. The a-helix. A. Polypeptide backbone showing the arrangements of the H-bonds. The N-H
of the peptide bond make an H-bond with the C=O of a peptide bond 3+ a bit residues further along
the chain. And this pattern repeats for every next peptide bond in the chain. B. Ribbon diagram with
the polypeptide backbone drawn in. C. The ribbon symbolizing the a-helix.
β-sheet
Fig.4.20 The anti-parallel b-sheet. (D) The protein backbone showing the H-bonds
between adjacent strains. (E) Ribbon diagram including the polypeptide backbone
(F) Symbolic representation of the b-sheet.
Antiparallel and parallel β-sheet
The energetically preferred
dihdreal angles are (φ, ψ) =
(–135°, 135°)
(φ, ψ) about (–140°, 135°)
(φ, ψ) about (–120°, 115°)
Anti-parallel β-sheet
The second most-frequently occurring element of secondary structure is the
antiparallel β-sheet. The N-H and C=O groups of a certain strand are hydrogen
bonded to C=O and N-H groups of adjacent chains that run parallel to it, but in the
opposite direction. The R-side groups in each strand alternately project above and
below the plane of the sheet (see fig 4.20)
Anti-parallel β-sheet example:
fibroin
Fig 4.21 Silk fibroin is organized in an anti-parallel b-sheet.
Proteins binds ‘active groups’, cofactors
Een voorbeeld: Photoactive Yellow Protein
blue-light sensor from H. halophila
Tyr42
Glu46
chromophore
Cys69
Thr50
Absorption of a blue-light photon triggers the photocycle
Experimental methods
• Site directed mutagenesis
P68  P68V, P68A, P68G
• Ultrafast spectroscopy of
WT and mutants (VIS and mid-IR)
• Molecular dynamics simulations
WT and mutants (ground state)
Simultaneous target analysis of all
samples
-Identical spectra
-Different dynamics
*wt fastest, p68v, p68a, p68g
-Different quantum yield
*wt most effective, p68v, p68a,p68g
Distributions of H-bonds with different strengths
Mid-IR:
t=0 spectra