Molecular and Cellular Biophysics

Introduction and Overview: Living Matter

Biomolecules: Structure, Function, Engineering

Cells: Structure, Function, Engineering
Energy Units
1 = 1.61019
1 = 40
1 = 4.1
3-4 kT (Energy molecules have due to thermal motion at body temperature)

Boltzmann-Maxwell Distribution (BMD)

Boltzmann factor determines the probability of being in a given state

Hyper structures: an intermediate level of organization

Hyper structures are large assemblies of different ions/ molecules/ macromolecules
that perform a function. They may be either quasi-equilibrium or non-equilibrium
(needing a flow of energy or material for their continued existence).

Two- state? cellular water

Mysterious Biophysical Phenomena

Galvanotropism
Magnetotropism
IR-Tropism
UV-Sensing& Emission
Bioenergetics

Thermodynamic Systems: Types

System - The matter within a defined region of space (reactants, products, solvents),
is the part in which we have special interest, it has definite boundaries, part of
universe chosen for study.

Surroundings The matter in the rest of the universe, is everything outside the
boundaries, the part of the universe that is not the system.
Open system Can exchange matter as well as energy with its surroundings
Closed system Can exchange energy with its surroundings. No transfer of matter
across the boundaries is possible
Isolated System Can exchange neither energy nor matter with its surroundings
Steady state Condition of an open system in which the rate of flow of energy or a
substance into the system is identical to the rate of flow out of the system.
Equilibrium Condition of no further net change in a close system, a system will be
in equilibrium if its internal energy is minimum.
Dynamic Equilibrium State of no net change, not no change, as in any quemical
equilibrium.

Properties of living matter

Excitability, Metabolism, Growth, Reproduction, Development, Inheritance

Biological Work and Energy

Heat - Thermal motion of molecules, energy of diffusive motion
Chemical bonds Enthalpy, ATP/Creatine/Pyruvate
Free Radicals molecules that have at least one unpaired electron
Electrical energy

Energy, Work and heat in organisms

The first Law - Statement of the conservation of energy, ways of heat exchange
The second Law Increase of entropy
Work Energy transfer by organized motion, involves the non- random movement
of particles, (expansion work, electrical work).
Heat Energy transfer by random motion, involves random movement of particles,
transfer of energy using thermal motion.

Heat Capacity (C) The heat energy required to raise the temperature of an object by
1C or 1K, extensive property but Specific heat capacity and Molar heat capacity are
intensive properties
= /
The energy of a system is a measure of its capacity to do work, the energy of a system
is the sum of the kinetic (movement energy of an object) and potential energies (stored
energy of an object).
= +
Reactions at constant volume = 0 , = , focus on heat.
The energy of a closed system can be changed by work done or by the system,
heat transfer across its boundaries
Thermodynamic potentials Internal Energy (U), Helmholtz energy (F= U-TS),
Enthalpy (U+pV), Gibbs Energy (G = U+pV-ST)

State variables
Path functions Functions governing transition between states (Heat, Work),
thermodynamic properties that relate to the preparation of the state.

State functions
A state function describes the state of a system, describes the current state of a system,
thermodynamic quantity whose value depends only on the current state of the system
and is independent of how the system has been prepared., how the system came to be
in that particular state is of no consequence.
Integral of a state function will be zero
Volume, Pressure, Temperature, Mass, Quantity, U, H, S, G

Exothermic/ Endothermic processes

Exothermic reaction One which involves the release of heat.
Endothermic reaction One which involves the absorption of heat.

Entropy and Probability

Is a measure of the degree of randomness or disorder of a system, thermodynamic

state function that is a measure of disorder, how things are arranged in space,
measures how close a system is to the state to corresponding no further change or
equilibrium.
=

=
Systems in equilibrium at temperature T have probability W of occupying a state i
with energy Ei.
Entropy change is often positive for spontaneous processes.

Enthalpy
The heat absorbed or emitted by a system at constant pressure, enthalpy is a state
function.
ENDO H

EXO

Activation Energy
Minimum energy input required to initiate a chemical reaction under given
conditions.

Gibbs Energy
Measure of spontaneity of a process, describes behavior of the system,
thermodynamic potential for a system under the constraints of constant temperature
and constant pressure.
=
For spontaneous process: < 0
Spontaneity Tendency of a chemical reaction to proceed in a certain direction
without the addition of energy.

Chemical Potential
Phase Space

Phase/State space All possible states that system can have

Hesss Law
Additivity of independently determined enthalpies; a statement of the first Law,
independent of the # of steps to the transformation, the total change in enthalpy will
be the same (number of steps is not important).

Phase transitions (1st and 2nd order)

Flux
Extensive and Intensive values
If two systems are joined (merged)
Extensive Sum Up (Mass, Volume, Energy) - The property is proportional to the
amount of material in the system, the sum of the properties of separate non-interacting
subsystems that compose the entire system.

Intensive Average (Temperature, Pressure) - Does not depend on the system size
or the amount of material in the system.

Idea of dissipation function

Respiratory chains
Life Potential
Stationary processes
Bio-molecular Forces
(Bonds and Interactions)

Bonds and Interactions

S-S bonds (Disulfide bridge) Is typically mostly in secreted proteins (there is no
free oxygen in the cell and consequently, no favorable oxidizing potential for S-S
bonding). Ex. Hair structures (strong links disulfide bonds)
A single covalent bond between the sulfur atoms to two amino acids called cysteine.
Electricity in Volume The Big Five
Electric PotentialElectric FieldCoulomb forceCurrent DensitySources and
SinksElectric Potential .

Covalent bonds: parameters

A covalent bond exists between two atoms if they share electrons between them to
form the 8 electron configuration, covalent bonds are important because they hold
together the back bones of all biological molecules.
Characterized in terms of energy.
Polarization Appears as result in differences of electronegativity, atoms with the
same or close electronegativity create non-polar or slightly polar bonds.
In a polar covalent bond the bond electrons are not distributed symmetrically along
the bond but they are shifted towards the more electronegative atom. The molecule
becomes a dipole.
Dipole Particle which has electric charges that are separated from each other in
space.
Dipole moment The measure of bond polarity is its dipole moment, vector quantity,
measure of the separation of positive and negative electrical charges in a system of
electric charges, that is, a measure of the charge system's overall polarity.
An object with an electric dipole moment is subject to a torque when placed in an
external electric field. The torque tends to align the dipole with the field. A dipole
aligned parallel to an electric field has lower potential energy than a dipole making
some angle with it
Polarizability Is the ability for a molecule to be polarized, polarizabilities determine
the dynamical response of a bound system to external fields, and provide insight into a
molecule's internal structure.
Water molecules are extremely polarizable

Angle When two or more atoms form covalent bonds with another central atom, these
bonds are oriented at precise angles to one another. The angles are determined by the
mutual repulsion of the outer electron orbitals of the central atom. These bond angles give
each molecule its characteristic shape

Electron Cloud Repulsion Theory (Valence Shell Electron Pair Repulsion, VSEPR)
is used to predict shapes and bond angles of simple molecules
Energy Breaking covalent bonds requires energy, and covalent bond formation
releases energy.

Short Range repulsion Atoms take space. If two atoms are forced together, they
push back. When two atoms approach each other at some distance the occupied
orbitals start to overlap, causing electrostatic repulsion. This repulsive force between
atoms acts over a very short range, but goes up sharply when that range is violated.
It prevents atoms from collapsing
Delocalization Electrons belonging to certain molecules are not attached to a
particular atom or bond in that molecule, they do not have a specific location (are not
localized); they cannot be drawn in a simple Lewis structure. The actual structure
with delocalized electrons is called a resonance hybrid.
Electrons become delocalized in order to stabilize a structure. For example the
benzene molecule.
Delocalized electron clouds, have a pool of electrons which they can share without
problems and not being damage, Delocalized electrons are spread across more than
one atom.

Resonance in conjugated systems and aromatic compounds, electron delocalization

lowers the potential energy of the substance and thus makes it more stable than any
of the contributing structures, important for Free Radical-Scavenging Effect.
Resonance Structures Peptide, Vitamin C, Guanosine

Polarizability and dipole moment

The ease with which electrons are shifted by an external electronic field is called
polarizability
In a molecule composed of atoms of various electronegativitys, the atoms with
lowest (smallest) electronegativitys hold partial positive charges and the atoms with
the greatest electronegativitys hold partial negative charges.
The extent of charge separation within a molecule is characterized by the dipole
moment . The dipole moment of a molecule is determined by the magnitudes of the
partial charges and by the distances between them.

Lehnard-Jones-Potential
ROS scavengers
Reactive oxygen species Are chemically reactive molecules containing oxygen.
Examples include peroxides, superoxide, hydroxyl radical, and singlet oxygen.

ROS are formed as a natural byproduct of the normal metabolism of oxygen and have
important roles in cell signaling and homeostasis. However, during times of
environmental stress (e.g., UV or heat exposure), ROS levels can increase
dramatically. This may result in significant damage to cell structures. Cumulatively,
this is known as oxidative stress. ROS are also generated by exogenous sources such
as ionizing radiation.
Hydrogen peroxide (H2O2), Hydroxyl radical (HO), Nitric oxide (NO), Peroxyl
radical (ROO), Peroxynitrite anion (ONOO), Singlet oxygen (1O2), Superoxide
anion (O2)

Van der Waals interactions

The phrase 'van der Waals interaction' has come to mean the adhesive/cohesive force
between molecules that are close together in space, the non-covalent associations
between molecules that arise from the electrostatic interactions among permanent
and/or induced dipoles.
Energy of the Van der Waals interaction depends on geometry. (Teflon)
Types of Van der Waals interactions- Ion/dipole, Ion/induced dipole, Dipole/dipole,
Dipole/dipole (hydrogen bonds), Dipole/induced dipole, dispersion forces
Dipole-dipole interactions depend on the orientations of the dipoles, can be attractive
or repulsive.
Favorable
Unfavorable
(-) (+)
(-)(-)
= = +
Dipole/Induced dipole interaction A molecule with a permanent dipole moment
creates a surrounding electrostatic field. This electrostatic field will shift the electrons
around a bit (induce a dipole moment) in a second molecule that is located nearby in
space. In liquid water (where molecules are close together), all water molecules are
polarized. The permanent dipole of each water molecule polarizes all adjacent water
molecules. The dipole of a water molecule induces change in the dipoles of all nearby
water molecule.

Hydrogen bonds
Despite being called a bond a hydrogen bond is weaker than the covalent bonds which
hold together organic molecules like proteins.

The electrostatic attraction between polar molecules that occurs when a hydrogen (H)
atom bound to a highly electronegative atom such as nitrogen (N), oxygen (O) or
fluorine (F) experiences attraction to some other nearby highly electronegative atom.
It is easy to make and reform hydrogen bonds without any need for energy inputs or
catalysis.
Types and strengths of hydrogen bonds
Two center short, straight, strong
Three center long, bent, weak
Four center long, bent, weak
Features Cooperativity, directionality, tenability, ubiquity.
In systems with multiple hydrogen bonds, the strength of one hydrogen bond is
increased by an adjacent hydrogen bond. Cooperativity of hydrogen bonding is
observed in base pairing and in folded proteins.
Directional bond, meaning that it is stronger when the hydrogen atom is aligned with
the two electronegative atoms.
Because of their directionality, tunability, and ubiquity in simple organic molecules
and biological polymers, hydrogen bonding interactions are one of nature's most
powerful devices of molecular recognition.
Hydrogen bonding also plays an important role in determining the three-dimensional
structures adopted by proteins and nucleic bases. In these macromolecules, bonding
between parts of the same macromolecule cause it to fold into a specific shape, which
helps determine the molecule's physiological or biochemical role.

Electrostatic Interactions (ionic)

Electrostatic interactions are between and among cations and anions, electrostatic
interactions dominate on the molecular scale and provide the fundamental basis for
all different bonded (ionic bonds) and non- bonded (ionic interactions) interactions.
Electrostatic interactions can be very strong, and fall off gradually with distance and
highly attenuated (dampened) by water.
In proteins positively charged with a negatively charged group interaction results in
a salt bridge (Ionic interactions between amino acids).

Salt bridges fall into the broader category of noncovalent interactions. A salt bridge
is actually a combination of two noncovalent interactions: hydrogen bonding and
electrostatic interactions, favorable electrostatic interactions between paired anionic
and cationic amino acid sidechains.

Screened electrostatic interactions (=Double electric layer forces)

The concept of the existence of the double layer at the surface of a metal being in
contact with an electrolyte appeared in 1879 (Helmholtz). That first theoretical model
assumed the presence of a compact layer of ions in contact with the charged metal
surface.
Two layers of opposite polarity form at the interface between electrode and
electrolyte. In 1853 he showed that an electrical double layer (DL) is essentially a
molecular dielectric and stores charge electrostatically. Below the electrolyte's
decomposition voltage the stored charge is linearly dependent on the voltage applied.

Hydrophobic Forces
Hydrophilic polar/polarizable
Hydrophobic no polar/ no polarizable
The hydrophobic effect is the observed tendency of nonpolar substances to aggregate
in aqueous solution and exclude water molecules. This occurs because interactions
between the hydrophobic molecules allow water molecules to bond more freely,
increasing the entropy of the system. The word hydrophobic literally means "waterfearing," and it describes the segregation and apparent repulsion between water and
nonpolar substances.
Very important in understanding a multitude of biological processes, protein folding
ligand-protein and protein-protein interactions, formation of micelles and bilayer
membrane, and insertion of membrane proteins into membranes.
Hydrophobic Interactions are important for the folding of proteins. This is important
in keeping a protein alive and biologically active, because it allow to the protein to
decrease in surface are and reduce the undesirable interactions with water.
The hydrophobic effect is responsible for the separation of a mixture of oil and water
into its two components. The hydrophobic effect is also responsible for the stability
of cell membranes, drives protein folding as well as the insertion of membrane
proteins into the nonpolar lipid environment and finally stabilizes protein-small
molecule interactions.

Unaggregated state Water population highly ordered, lower entropy, energetically

unfavorable.
Aggregated state Water population less ordered, higher entropy, energetically more
favorable

Chaotropes and Cosmotropes

Chaotropes Destroy hydrogen bonds.
Cosmotropes Facilitate creation of new hydrogen bonds.

Superhydrophobicity
Omniphobicity
Measuring hydrophobicity
Contact angle and surface tension
The contact angle is the angle, conventionally measured through the liquid, where a
liquid/vapor interface meets a solid surface. It quantifies the wettability of a solid
surface by a liquid via the Young equation. If angle > 90 = hydrophobic.
The hydrophobic effect can be easily studied through changes in the surface tension
for an air/water interface.

LogP and MHP as a reference value

MHP Molecular hydrophobic potential
LogP Distribution coefficient water (octanol), measure concentration in octanol.

Capillary Forces
Capillary action is the ability of a liquid to flow in narrow spaces without the
assistance of, and in opposition to, external forces like gravity.

Hydrodynamic Forces
Evaluating shear forces close to the surface

Depletion Forces
Restricted in their motion system tries to minimize the area.

Depletion forces are often regarded as entropic forces, as was first explained by the
established Asakura-Oosawa model. In this theory the depletion force arises from an
increase in osmotic pressure of the surrounding solution when colloidal particles get
close enough such that the excluded cosolutes (depletants) cannot fit in between them

Brush (steric) forces

Steric effects arise from the fact that each atom within a molecule occupies a certain
amount of space. If atoms are brought too close together, there is an associated cost in
energy due to overlapping electron clouds (Pauli or Born repulsion), and this may affect
the molecule's preferred shape (conformation) and reactivity.

For polymers with large molecular weights, the chain dimensions are similar to the
range of van der waals attraction. This means that at long as they can generate
repulsion, they can generate stability. This stabilization is created by attaching
macromolecules to the surface of the particle as shown below.

Solvation (hydration) forces

When two surfaces or particles approach closer than a few nanometers, continuum
theories of van der Waals and double layer forces often fail to describe their
interaction.
Solvation forces depend not only on the properties of the intervening medium but also
on the chemical and physical properties of the surfaces.
The short-distance interactions are usually referred to as solvation forces, structural
forces, or when the medium is water hydration forces.

Undulation forces

Methods for studying bio molecular interactions

Affinity chromatography, equilibrium dialysis, surface plasmon resonance,
isothermal titration calorimetry.
Thermal Analysis

What TA can tell you

Glass Transitions, Melting and boiling points, crystallization time and temperature,
specific heat, reaction kinetics, purity
Calorimeter measures the heat into or out of a sample
Differential calorimeter Measures the heat of a sample relative to a reference
Differential scanning calorimeter Does all of the above and heats the sample with
a linear temperature ramp.
Endothermic Heat flows into the sample
Exothermic Heat flows out of the sample
Heat Flux Principle The differential temperature between the sample and reference
is converted to differential heat flow in a way that is analogous to current flow in
Ohms Law.

Isothermal Titration Calorimetry

A single experiment is sufficient to obtain all of the thermodynamic components.
The amount of power required to maintain a constant temperature difference between
the reaction cell and the reference cell is measured.
Exothermic reaction = negative peak
Endothermic reaction = positive peak
Heat absorbed or generated during titration directly proportional to amount of bound
ligand.

Differential Scanning Calorimetry

Measures the temperatures and heat flows associated with transitions in materials as
a function of time and temperature in a controlled atmosphere.
This measures provide quantitative and qualitative information about physical and
chemical changes that involve endo or exo processes, or changes in heat capacity.
Calculation of all the thermodynamic parameters characterizing a biological
molecule.
Biopolymers:

Molecular Dynamics, structure and folding

Amino acids: groups

Isoelectric point
Point in the ph where its charge is zero, pH at which the net charge on a
macromolecule is zero.

Hierarchy in protein organization

Primary structure Stabilized by covalent bonds, sequence of amino acids, which
form a chain connected by peptide bonds, the amino acids sequence of a protein
determines the higher levels of structure of the molecule.
Secondary structure Stabilized by hydrogen bonds, Alpha helix, Beta pleated
Tertiary structure Is the way random coils, alpha-helixes and beta pleated sheets
fold in respect to each other, amino acids which are very distant in the primary
structure might be close in tertiary one because the folding of the chain. Hydrophobic
interactions, electrostatic interactions, Van der Waals.
Quaternary structure Polypeptide chains with covalent bonds, Human insulin

Kuhn Segments
Is a theoretical treatment, developed by Werner Kuhn, in which a polymer chain is
considered as a collection of N Kuhn segments each with a Kuhn length b. Each Kuhn
segment can be thought of as if they are freely jointed with each other. Each segment
in a freely jointed chain can randomly orient in any direction without the influence of
any forces, independent of the directions taken by other segments.
Macromolecules as random Walks
Kuhn considered an equivalent ideal chain with N connected segments, now called
Kuhn segments that can orient in any random direction.

Ramachandran plot
Finding minimal energy as combination of angles.

Ramachandran used computer models of small polypeptides to systematically vary

phi and psi with the objective of finding stable conformations.
Is a way to visualize backbone dihedral angles against of amino acid residues in
protein structure.

Protein contact map

Structure interpretable, plot amino acids only know the order not names, two times
plotting the numbers of amino acids, which amino acids have contact.
A protein contact map represents the distance between all possible amino acid residue
pairs of a three-dimensional protein structure using a binary two-dimensional matrix.
Protein contact map describes the pairwise spatial and functional relationship of
residues in a protein and contains key information for protein 3D structure prediction.

Motif
Certain group of amino acids close to each other
Sequence motif Always have functional implications, finding sequence motifs
identification.
In proteins, a structural motif describes the connectivity between secondary structural
elements.

The sequences of many proteins contain short, conserved motifs that are involved in
recognition and targeting activities, often separate from other functional properties of
the molecule in which they occur. These motifs are linear, in the sense that threedimensional organization is not required to bring distant segments of the molecule
together to make the recognizable unit. The conservation of these motifs varies: some
are highly conserved while others, for example, allow substitutions that retain only a
certain pattern of charge across the motif.
Functional motif Sequence or structural motif that is always associated with a
particular biochemical function

Domain
Protein folding
Process in which a polypeptide chain goes from a linear chain of amino acids with
vast number of more or less random conformations in solution to the native, folded
tertiary(and for multichain proteins, quaternary) structure.

Protein folding is the process by which a protein structure assumes its functional
shape or conformation.
Enthalpy, main driving force of protein folding.
Thermodynamically protein folding is a spontaneous process
Protein structures are free-energy minimizers
Negative G- decrease in free energy for a process (favorable)
Reaction would go spontaneous in that direction.
Change in enthalpy reflects number and kinds of chemical bonds in reactants and
products, making bonds/interactions gives negative H (favorable).
Increase in disorder gives positive S (favorable).
Proteins fold spontaneously under physiological conditions
Native state Folded, biologically functional

Protein misfolding
Denaturation and renaturation
Denatured state Unfolded or partially folded
Proteins can be denatured in vitro by chemical agents like, urea, or extremes of heat
or pH, and then refolded by diluting out the chemical denaturant, changing pH,etc.
Proteins fold on a defined path way.
Renaturing of proteins difficult due to crowded environment.
High level of energy, unfolded state.

Chaperones/Chaperonins
Chaperons Helps proteins to fold, provides isolated space for new proteins to fold

Protein p53
Contains both ordered and disordered domains.

Central guard of our cell, central position on keeping our cells not malignant, launches
self-killing process to control cell, it can bind/interact/inactivate proteins, flexible and
adaptable, unique structure functions.
Is crucial in multicellular organisms, where it prevents cancer formation, thus,
functions as a tumor suppressor. As such, p53 has been described as "the guardian of
the genome" because of its role in conserving stability by preventing genome
mutation.

Natively unfolded proteins

Proteins who have little or no ordered structure under physiologic conditions, low
content of hydrophobic amino acids residues.

Mercaptoethanol
Urea
Denaturing agent, destroys effectively hydrogen bonds, disrupts the non-covalent
bonds within the protein that stabilize its native tertiary and quaternary structure.

Degeneracy and elasticity

Entropy determines the elastic properties of polymer chains

Elastin
Its a rubber like material, an important load-bearing protein, used in places where
mechanical energy is required to be stored as deformation, particularly with cyclic
loading (arteries, lungs some ligaments).
Requires Lys-cross linking
Elastin fibers must expand and contract over a lifetime, and can be extended nearly
three times their resting length without damaging their flexibility.
As it is stretched, the chain is moving from a more probable (higher entropy) to a less
probable (low entropy) state.
It is this lowering of entropy of the conformation that causes the retractive force, so
rubber like elastin is described as an entropy spring.

Sickle cell disease

The term sickle cell disease (SCD) describes a group of inherited red blood cell
disorders. People with SCD have abnormal hemoglobin, called hemoglobin S or
sickle hemoglobin, in their red blood cells.
Sickle-shaped cells are not flexible and can stick to vessel walls, causing a blockage
that slows or stops the flow of blood. When this happens, oxygen cant reach nearby
tissues.

Some applications in cell biology

Bacterial Genome as Random Walk example The overwhelming probability that R
is close to zero is responsible for the elastic response of polymer chains due to an
applied load.
In-Vivo Conformation (DNA or proteins) Fluorescent marker
Scaling of protein size as function of the number of amino acids residues

Folding diseases
Many diseases are the result of defects in protein folding.
Cystic fibrosis
Abnormal protein aggregationprion (abnormal protein) diseases, Alzheimer
disease, Parkinson disease, Huntington disease

Alzheimer disease mechanisms

DNA & RNA Biophysics:

Shape and Transformations
Dynamics and Function
Epigenetics

Structural levels of DNA/ RNA

DNA folding (interactions)
DNA supercoiling
DNA-bending: Wedge Models vs Junction Models
DNA physical properties
DNA origami/ engineering
A-, B- and Z- families of DNA
DNA-histone Interaction

Repressosome
Biophysical aspects of epigenetic regulation
Hypercromisity phenomenon
RNA versatility
Techniques for studying biomolecules
Spectroscopy
Circular Dichroism
Light Scattering

Polarization of light
Circular polarization
Elliptical polarization
Circular dichroism
Optical activity
Ellipticity
Dn/ dc parameter
Rayleigh Scattering
Mie Scattering
Raman Scattering
Static light scattering
Dynamic light scattering
Autocorrelation function
Interaction of light with matter
Transmittance and Adsorption
Lambert-Beer law
Far-, mid- and near-infrared