PRIMER ON

BIOMOLECULES,
PROTEINS AND CELLS
Lecture 7
PHY2325
The Guts of the Cell
Before we look at
biological examples
of motion lets recap
what is in a cell,
what the guts are
made of, what the
cell is made of.
Whats it Like Inside E Coli?
David Goodsell - E Coli

Medium and big molecules (26%):

~15% Proteins
~6% RNA,
~1% DNA
~2% Lipids,
~2% polysaccharides

Small molecules (74%): ~70% water, ~1.2% ions, ~1% sugars,

~1% fatty acids, ~0.4% ind. nt, ~0.4% ind. amino acids.
How do we look into a cell?
Fluorescent Proteins

Green Fluorescent Protein (GFP)

Discovered and isolated from jellyfish

in the 1960s

Easily used to tag proteins expressed

in living cells

Nobel Prize in 2008

Enhanced GFP (EGFP) was developed

for use with standard fluorescent filter
cubes and laser lines
Fluorescent Proteins

There are now many different GFP variants covering the visible spectrum. Many developed by
Tsien through directed evolution.

Can develop DNA plasmids which encode for your protein of interest tagged with an FP

Very important for physical measurements on living cells

2008 Nobel Osamu Shimomura, Martin Chalfie, Roger Y. Tsien, "for the discovery and
development of the green fluorescent protein, GFP".
Transfections - Plasmid Design
So how does one use GFP? - Design a circular DNA plasmid
(typically <10kb) which contains the GFP sequence and the
sequence of the protein of interest.

Mammalian
and
bacterial
antibiotic
resistance
genes
Fluorescence Imaging in Biophysics

Mechanics of the
Cytoskeleton

Forces, Viscous Relaxation

The E Coli Ruler

AFM

EM

0.5 -

1-
Structures we may be interested in ....

Biological structures we will see in

this course cover a huge range of
length scales.

In this case biomolecules (10-10 m)

up to E. Coli (10-6 m) cover 4
orders of magnitude!
Structures we may be interested in ...

Biological structures we will see in

this course cover a huge range of
length scales.

In this case, E. Coli (10-6 m) up to

a tissue (10-3 m) cover 3 orders of
magnitude!

In total we will study physical

phenomena over 7 orders of
magnitude!
Molecular Assemblies
The cell is ultimately a collection of proteins, lipids, carbohydrates
and nucleic acids which form functional assemblies
DNA and RNA are Polymers of Nucleic Acids

Nucleotides are the monomers that make up the genetic polymers

DNA (deoxy-ribo-nucleic acid) and RNA (ribo-nucleic acid)

4 type bases: cytosine (C), guanine (G), adenine (A), thymine (T) in
DNA and uracil (U) in RNA

Role of DNA: to store genetic information

Role of RNA: controls protein expression
DNA and RNA are Polymers of Nucleic Acids

DNA can be replicated

The Central Dogma
Protein Structure Hierarchy
Proteins are Polymers of Amino Acids
Proteins are made of many amino acids (>50)

Short chains of fragments are called: peptides

Each amino acid shares a backbone structure

amino group carboxyl group

direction

The side chain - R -distinguishes the diff. amino acids (20).

Sequence of amino acids gives rise to structure and

function of proteins (1o - primary structure)
Proteins are Polymers of Amino Acids
Once assembled by a ribosome, the protein can fold,
based on the distribution of charge and steric interactions.

Common folding patterns include:

alpha-helices (single spring like structure)
beta-sheets (mostly flat loops)

These patterns are referred to as (2o) secondary structure -

as a results of hydrogen bonds.

The global folding pattern of the entire protein is referred to

as the (3o) tertiary structure. Association of proteins is
termed (4o) quaternary structure.
Secondary Structure
The Alpha-Helix
In the -helix, the carbonyl oxygen of
residue i forms a hydrogen bond
with the amide of residue i+4.

Although each hydrogen bond is

relatively weak in isolation, the sum of
the hydrogen bonds in a helix makes
it quite stable.

The propensity of a peptide for

forming an -helix also depends on its
sequence.

Cartoon
Representation
The Beta-Sheet

In a -sheet, carbonyl oxygens and amides

form hydrogen bonds.

These secondary structures can be either

antiparallel (as shown) or parallel and need not
be planar (as shown) but can be twisted.

The propensity of a peptide for forming -

sheet also depends on its sequence.

The -barrel of GFP

Polar Groups Localization

Polar/Non-Polar R group localization is central to protein structure/

function wether they are found in an aqueous environment or in the cell
membrane, or even spanning part or all of the cell membrane.
Nonpolar Groups Localization

Nonpolar R groups tend

to be found buried inside
protein structure

Myoglobin
Blue = non-polar
R-group

Red = Heme
Fatty Acids store energy but also form Structure (Cell
Membrane)

Cell Membrane is composed

of phospholipids with polar
heads and non-polar tails.
Hydrophilicity and
hydrophobicity really matter.
Trans-membrane Proteins

Nonpolar R groups tend to be found on the surface of

membrane domains.

Polar R groups tend to be found on the surface of non-

membrane bound domains.

Energetic cost to having polar R-groups in nonpolar

environments and vice versa.

Polar
Nonpolar
Tertiary Structure
Tertiary Structure

random coil

-helix

-sheet
Tertiary Structures Can Vary Widely!

The function determines the form!

Quaternary Structure
Higher Order Assembly
Tetramer Hexamer Filament

Hemoglobin
DNA Helicase
Actin Cytoskeleton

4 subunits each 6 subunits for Monomers (G-actin)

containing a Fe heme separating dsDNA in bind ATP and
group for O2 ssDNA.
polymerize into a
transport. The H-bonds in DNA are double stranded
dimers rotate 15 broken using the filament (F-actin).
upon binding O2. energy from ATP Ubiquitous protein in
hydrolysis nature.
Classes of Proteins

Functional definition:
Enzymes: Accelerate biochemical reactions
Structural: Form biological structures
Transport: Carry biochemically important substances
Defence: Protect the body from foreign invaders

Structural definition:
Globular: Complex folds, irregularly shaped tertiary structures
Fibrous: Extended, simple folds -- generally structural proteins

Cellular localization definition:

Membrane: In direct physical contact with a membrane; generally
water insoluble.
Soluble: Water soluble; can be anywhere in the cell.
Filaments in the Cell
pellinglab.net
The Cytoskeleton
Actin
Microtubules
Intermediate Filaments
The Cytoskeleton
How Do Proteins Fold?
Anfinsens Experiment - 1961
Structural Determinant - Entropy

G = H T S
Structural Determinant - Chemistry
Unassisted Protein Folding is Rarely Linear
Complex energy landscape
during protein folding

We will discuss the influence of

mechanical force on folding
pathways
Can Physics Alone Predict Protein Folding?

Proteins involved in folding other proteins exert mechanical and chemical forces which
drastically alter the folding energy landscape
Motor Proteins
Types of Motor Proteins
Motor Proteins are associated with the Actin and Microtubule
cytoskeletal filaments
Motor Protein Functions
Molecular Machines FITB!

Kinesin Car Engine

Size

Fuel

Speed

Efficiency
Imaging Molecular Motors

Kodera et al. Nature 468, 72 (2010)

RNA Polymerase

Nature 426, 684 (2003)

Kinesin - Hand over Hand
The Flagella

Rotational motion to
drive motility.

Self-organizing

Curvature of the filament

Mitochondria

Conversion of glucose is required to setup an electrochemical gradient

between the mitochondrial matrix and the inter membrane space.

Required for ATP production.

C6H12O6 (aq) + 6 O2 (g) 6 CO2 (g) + 6 H2O (l)

ATP Synthase
} Inter-membrane space
(H+ rich)

} Inner membrane

} Matrix
ATP Synthase
ATP Synthase
Cellular respiration results in a H+
gradient.

ATP Synthase utilizes the gradient

to drive rotation of the F0
subunit.

The F1 subunit is mechanically

coupled to F0 and rotates as well.

Rotation of the axle results in

conformational changes in the F0
unit, catalyzing the formation of
ATP.

Mechanical signalling drives the

conversion of of ADP+Pi to ATP!
ATP Synthase

Observation of F1-ATPase counter clockwise rotation

Nature 386, 299 (1997)

Hybrid Bio-Inorganic nano propellor

nanopropeller length 750-1400 nm, diameter 150 nm.

Science 290, 1555 (2000)

Direct Observation of F1 Rotation
High speed atomic force microscopy (AFM) has been
employed to directly image the rotation of F1 ATPase

No ATP

With ATP

Science 333, 755 (2011)

Direct Observation of F1 Thermal Fluctuations

Thermally driven movements and fluctuations

No ATP

10 frame/sec, 18x15 nm2

Science 333, 755 (2011)

Direct Observation of F1 Rotation with ATP

2uM ATP 4uM ATP

Higher ATP concentrations results in more activity.

10 frame/sec, 17x12 nm2

Science 333, 755 (2011)

Direct Observation of F1 Rotation

Tracking the brightest pixel reveals counter clockwise rotation.

DIRECT observation of rotational motion and thermal fluctuations

2uM ATP

Science 333, 755 (2011)

Direct Observation of F1 Rotation

Counter clockwise
motion is preferred.

Full rotations taking

place over ~2sec

Science 333, 755 (2011)