Molecular Biophysics

12824 BCHS 6297Lecturers 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

Keelerhttp://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 bondThe 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 | + -

CH3OH H—C—OH C O | H

or even stronger polarity H + - + -

H C O C O

O> N> C, H electronegativity - + + - + -

O H C N C O

Geometry also determines polarity

• + - • while C Cl is polar

carbon tetrachloride is not. The sum of the vectors equals zero and it is therefore a nonpolar molecule

CCl4 = 1+2+3+4 = 0

CCl Cl

Cl Cl1

2

3

4

CCl Cl

Cl

2

3

4 H

CHCl3 is polar

Electrostatic interactions

by coulombs law F= kq1q2 q are charges r2D r is radiusD = dielectric of the media, a shielding of charge. And k =8.99 x109Jm/C2

D = 1 in a vacuumD = 2-3 in greaseD = 80 in water

Responsible for ionic bonds, salt bridges or ion pairs,optimal electrostatic attraction is 2.8Å

Dielectric effect Dhexane 1.9benzene 2.3diethyl ether 4.3CHCl3 5.1acetone 21.4Ethanol 24methanol 33H2O 80HCN 116

H2O is an excellent solvent and dissolves a large array of polar molecules.

However, it also weakens ionic and hydrogen bondsTherefore, biological systems sometimes exclude H2O to

form maximal strength bonds!!

Hydrogen bonds

O-H N N-H O 2.88 Å 3.04 Å

H bond donor or an H bond acceptor

N H O C

3-7 kcal/mole or 12-28 kJ/molevery 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

ÅH 1.2 1.0 kcal/molC 2.0 4.1 kJ/molN 1.5 weak interactionsO 1.4 important when many atomsS 1.85 come in contactP 1.9

Can only happen if shapes of molecules match

Hydrophobic interactionsNon-polar groups cluster together

G = H - TS

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 WATERA cage of water molecules surrounding the non-polar molecule

This cage has more structure than the surrounding bulk media.

G = H -TS

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

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

Process H -TS G

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

G = H -TS driven by TS

Amino Acids:The building blocks of proteins

amino acids because of the carboxylic and amino groupspK1 and pK2 respectively pKR is for R group pK’s

pK1 2.2 while pK2 9.4

pK1pK2

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

polypeptides

Proteins are molecules that consist of one or more polypeptide chains

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 AcidsThere are three main physical categories to describe amino acids:

1) Non polar “hydrophobic” nine in allGlycine, Alanine, Valine, Leucine, Isoleucine,

Methionine, Proline, Phenylalanine and Tryptophan

2) Uncharged polar, six in allSerine, Threonine, Asparagine, Glutamine Tyrosine,

Cysteine

3) Charged polar, five in allLysine, Arginine, Glutamic acid, Aspartic acid, and

Histidine

Amino Acids You must know:

Their namesTheir structureTheir three letter codeTheir one letter code

H2N CH C

CH2

OH

O

OH

Tyrosine, Tyr, Y, aromatic, hydroxyl

Cystine consists of two disulfide-linked cysteine residues

Acid - Base properties of amino acids

[HA]][Alog pK pH

-

ji pKpK21 pI

Isoelectric point: the pH where a protein carries no net electrical charge

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.

ionconcentratlength x path (degrees)roration observed ][ 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 C This works for all amino acids.

An example of an amino acid with two asymmetric carbons

Structural hierarchy in proteins

Color conventions

Protein GeometryCORN 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 / backbone rotamers

Regular Secondary Structure Pauling and Corey

Helix Sheet

alpha helix

Properties of the helix

• 3.6 amino acids per turn• Pitch of 5.4 Å• O(i) to N(i+4) hydrogen bonding• Helix dipole• Negative and angles, • Typically = -60 º and = -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 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 () 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 Beta-Sheets

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 beta-bulges) and greater exposure to solvent.