Transcript Structural Genomics - University of Houston

Molecular Biophysics
12824 BCHS 6297
Lecturers held Tuesday and Thursday
10 AM – 12 Noon 402B-HSC
Aims of Course
• Understand role of physics in protein
structure
• Overview of the Electronic Spectroscopies
• Understand the application of kinetics and
thermodynamics to study enzyme catalysis
and protein folding
• Basics of NMR and X-ray crystallography
Suggested texts
• Principles of Physical Biochemistry, van Holde
• Structure and Mechanism in Protein Science,
Fersht
• On-line resourses
• Understanding NMR spectroscopy, by James
Keeler
http://www-keeler.ch.cam.ac.uk/lectures/
• Principles of Protein Structure Using the Internet,
http://www.cryst.bbk.ac.uk/PPS2/course/index.html
Structural Biology of the HIV proteome
Molecular Forces in Protein Structure
•
•
•
•
•
•
•
•
Interactions, forces and energies
Covalent Interactions
Non-bonded Interactions
Electrostatic interactions: salt bridges, hydrogen
bonds, partial charges and induction
The Lennard-Jones potential and van der Waals
Radii
The effect of solvent and hydrophobic interactions
Dielectric effects
The hydrophobic effect
Covalent bond
The force holding two atoms together by the sharing of a pair of
electrons.
H + H  H:H or H-H
The force: Attraction between two positively charged nuclei and a
pair of negatively charged electrons.
Orbital: a space where electrons move around.
Electron can act as a wave, with a frequency, and putting a
standing wave around a sphere yields only discrete areas by which
the wave will be in phase all around. i.e different orbitals.
Polarity of Bonds
H
d+ d-
|
CH3OH H—C—OH
|
H
H
H
d-
d+
O H
d+
d-
C
O
C O
or even stronger polarity
d+ d-
C O
O> N> C, H electronegativity
d+ d-
C N
d+
d-
C O
Geometry also determines polarity
•
d+
d• while C
Cl is polar
carbon tetrachloride is
not. The sum of the
vectors equals zero
and it is therefore a
nonpolar molecule
mCCl4 = m1+m2+m3+m4 = 0
Cl
Cl
m4
m3
m1
C
Cl
Cl
m2
Cl
m3
Cl
H
m4
C
m2
Cl
CHCl3 is polar
Electrostatic interactions
by coulombs law F= kq1q2
r2D
q are charges
r is radius
D = dielectric of the media, a shielding of charge.
And k =8.99 x109Jm/C2
D = 1 in a vacuum
D = 2-3 in grease
D = 80 in water
Responsible for ionic bonds, salt bridges or ion
pairs,optimal electrostatic attraction is 2.8Å
Dielectric effect
hexane
benzene
diethyl ether
CHCl3
acetone
Ethanol
methanol
H2O
HCN
D
1.9
2.3
4.3
5.1
21.4
24
33
80
116
H2O is an excellent solvent and dissolves a large array of
polar molecules.
However, it also weakens ionic and hydrogen bonds
Therefore, biological systems sometimes exclude H2O to
form maximal strength bonds!!
Hydrogen bonds
O-H
N
2.88 Å
N-H
O
3.04 Å
H bond donor or an H bond acceptor
N H
O C
3-7 kcal/mole or 12-28 kJ/mole
very strong angle dependence
A hydrogen bond between two
water molecules
.
van der Waals attraction
Non-specific attractions 3-4 Å in distance (dipole-dipole attractions)
Contact Distance
H
C
N
O
S
P
Å
1.2
2.0
1.5
1.4
1.85
1.9
1.0 kcal/mol
4.1 kJ/mol
weak interactions
important when many atoms
come in contact
Can only happen if shapes of molecules match
Hydrophobic interactions
Non-polar groups cluster together
DG = DH - TDS
The most important parameter for determining a biomolecule’s shape!!!
Entropy order-disorder. Nature prefers to maximize entropy “maximum
disorder”.
Enthalpy How can structures form if they are unstable?
Structures are driven by the molecular interactions of
the water!
STRUCTURED WATER
A cage of water molecules surrounding the non-polar molecule
This cage has more structure than the surrounding bulk media.
DG = DH -TDS
Entropy decreases!! Not favorable! Nature needs to be more
disorganized. A driving force.
SO
To minimize the structure of water the hydrophobic molecules
cluster together minimizing the surface area. Thus water is
more disordered but as a consequence the hydrophobic
molecules become ordered!!!
Proton and hydroxide mobility is large
compared to other ions
• H3O+ : 362.4 x 10-5 cm2•V-1•s-1
• Na+:
51.9 x 10-5
• Hydronium ion migration; hops by switching
partners at 1012 per second.
Free energy of transfer for hydrocarbons
form water to organic solvent
DH
Process
-TDS
DG
CH4 in H2O 
CH4 in C6H6
11.7
-22.6
-10.9
CH4 in H2O 
CH4 in CCl4
10.5
-22.6
-12.1
C2H6 in H2O  C2H6 in C6H6
9.2
-25.1
-15.9
Amphiphiles form micelles, membrane
bilayes and vesicles
• A single amphiphile is surrounded by water, which forms
structured “cage” water. To minimize the highly ordered
state of water the amphiphile is forced into a structure to
maximize entropy
DG = DH -TDS driven by TDS
Amino Acids:
The building blocks of proteins
pK2
pK1
a amino acids because of the a carboxylic and a amino groups
pK1 and pK2 respectively pKR is for R group pK’s
pK1  2.2 while pK2  9.4
In the physiological pH range, both carboxylic and
amino groups are completely ionized
Amino acids are Ampholytes
They can act as either an acid or a base
They are Zwitterions or molecules that have both
a positive and a negative charge
Because of their ionic nature they have
extremely high melting temperatures
Amino acids can form peptide
bonds
Amino acid residue
peptide units
dipeptides
tripeptides
oligopeptides
Proteins are
molecules that
consist of one or
more
polypeptide
chains
polypeptides
Peptides are linear polymers that range from 8 to 4000
amino acid residues
There are twenty (20) different naturally occurring
amino acids
Characteristics of Amino Acids
There are three main physical categories to describe amino
acids:
1) Non polar “hydrophobic” nine in all
Glycine, Alanine, Valine, Leucine, Isoleucine,
Methionine, Proline, Phenylalanine and Tryptophan
2) Uncharged polar, six in all
Serine, Threonine, Asparagine, Glutamine Tyrosine,
Cysteine
3) Charged polar, five in all
Lysine, Arginine, Glutamic acid, Aspartic acid, and
Histidine
Amino Acids
You must know:
Their names
Their structure
Their three letter code
Their one letter code
O
H2N
CH
C
OH
CH2
Tyrosine, Tyr, Y, aromatic, hydroxyl
OH
Cystine consists of two disulfide-linked
cysteine residues
Acid - Base properties of amino acids
 [A- ] 

pH  pK  log
 [HA]
Isoelectric point: the pH where
a protein carries no net
electrical charge
1
pI  pK i  pK j 
2
For a mono amino-mono carboxylic
residue pKi = pK1 and pKj = pK2 ; for
D and E, pKi = pK1 and pKj - pKR ;
For R, H and K, pKi = KR and pKj =
pK2
The tetra peptide Ala-Tyr-Asp-Gly or AYDG
Greek lettering used to identify atoms in lysine or glutamate
Optical activity - The ability to rotate plane - polarized
light
Asymmetric carbon atom
Chirality - Not superimposable
Mirror image - enantiomers
(+) Dextrorotatory - right - clockwise
(-) Levorotatory - left counterclockwise
}
Na D Line passed through polarizing filters.
observedroration(degrees)
[a ] 
pathlength xconcentration
25
D
Operational
definition only
cannot predict
absolute
configurations
The Fischer Convention
Absolute configuration about an asymmetric carbon
related to glyceraldehyde
(+) = D-Glyceraldehyde
(-) = L-Glyceraldehyde
In the Fischer projection all bonds in the horizontal
direction is coming out of the plane if the paper,
while the vertical bonds project behind the plane of
the paper
All naturally occurring amino acids that make up
proteins are in the L conformation
The CORN method for L
isomers: put the hydrogen
towards you and read off
CO R N clockwise
around the Ca This works
for all amino acids.
An example of an amino acid with two
asymmetric carbons
Structural hierarchy in proteins
Color conventions
Protein Geometry
CORN LAW amino acid with L configuration
Greek alphabet
The Polypeptide Chain
Polypeptide geometry
• Pauling and Corey
Peptide bond
• C-N bond displays partial double bond character
Peptide bonds generally adopt a trans
configuration
Peptide Torsion Angles
Torsion angles determine flexibility of backbone structure
Steric hindrance limits backbone flexibility
Rammachandran plot for L amino acids
Indicates energetically favorable f/y backbone rotamers
Regular Secondary Structure
Pauling and Corey
Helix
Sheet
alpha helix
Properties of the a helix
•
•
•
•
•
•
3.6 amino acids per turn
Pitch of 5.4 Å
O(i) to N(i+4) hydrogen bonding
Helix dipole
Negative f and y angles,
Typically f = -60 º and y = -50 º
Distortions of alpha-helices
• The packing of buried helices against other
secondary structure elements in the core of the
protein.
• Proline residues induce distortions of around 20
degrees in the direction of the helix axis. (causes
two H-bonds in the helix to be broken)
• Solvent. Exposed helices are often bent away from
the solvent region. This is because the exposed
C=O groups tend to point towards solvent to
maximize their H-bonding capacity
Top view along helix axis
Helical bundle
310 helix
•
•
•
•
Three residues per turn
O(i) to N(i+3) hydrogen bonding
Less stable & favorable sidechain packing
Short & often found at the end of a helices
Helical propensity
Peptide helicity prediction
• AGADIR
http://www.embl-heidelberg.de/Services/serrano/agadir/agadir-start.html
Agadir predicts the helical behaviour of
monomeric peptides
It only considers short range interactions
beta (b) sheet
• Extended zig-zag
conformation
• Axial distance 3.5 Å
• 2 residues per repeat
• 7 Å pitch
Antiparallel beta sheet
Antiparallel beta sheet side view
Parallel beta sheet
Parallel, Antiparallel and Mixed BetaSheets
Beta sheets are twisted
• Parallel sheets are less twisted than antiparallel and are always buried.
• In contrast, antiparallel sheets can withstand greater distortions (twisting and betabulges) and greater exposure to solvent.