Information Storage and Processing in Biological Systems: A seminar - - PowerPoint PPT Presentation

Information Storage and Processing in Biological Systems: A seminar course for the Natural Sciences

• Sept. 11

Biological Information, Sept 16 DNA, Gene regulation Sept 18 Translation and Proteins

Sept 23 Enzymes and Signal transduction

Sept 25 Biochemical Networks Sept 30 Simple Genetic Networks (Dr. Jacob) Oct 2

C - Introduction to Proteins / Protein Functions

Proteins carry out a wide variety of functions in, on and outside the cell. For the purpose of this course, we will generalize these functions into three

• categories. These are not mutually exclusive and many proteins fit into more

than one of these categories. 1 - Structural 2 - Enzymatic 3 - Signal Transduction (information processing)

C1 - Protein Functions: Structural

Proteins can form large complexes that function primarily as structural elements: Protein coats of viruses. These are large, regular repeating structures composed of 100-1000’s of protein subunits. (Figs 6-74 and 6-72, Alberts).

Electron micrographs of A) Phage T4, B) potato virus X, C) adenovirus, D) influenza virus. SV40 structure determined by X-ray crystallography.

Cytoskeleton in eukaryotic cells is responsible not only for determining shape but also in cell movement, mechanical sensing, intracellular trafficking and cell division. A human cell grown in tissue culture and stained for protein (such that only large regular structures are highlighted). Note the variety of structures (Fig 16-1, Alberts)

Microtubules form by the polymerization of tubulin subunits. Whether the polymer grows or shrinks is influenced conditions in the cell - Dynamic Instability

(Fig 16-33, Alberts; for discussion of dynamic instability see Flyvbjerg H, Holy TE, Leibler S. Stochastic dynamics of microtubules: A model for caps and catastrophes. Phys Rev Lett. 1994 Oct 24;73(17):2372-2375.

C2 - Protein Functions: Enzymatic

Enzyme: a protein* that catalyzes a chemical reaction, where a catalyst is defined as a substance that accelerates a chemical reaction without itself undergoing change.

* some RNA molecules can also be considered enzymes

A A + B B C + D X Y

• Specificity
• Accelerated reaction rates
• Control (regulation)
• Enzymes can only affect the rate (kinetics) of a reaction, they can not

make a reaction more energetically favorable.

• Enzymes can be saturated by substrate.

Basics of Enzyme Kinetics v = Vs (KM + s)

Michaelis-Menton Kinetics - for a simple enzyme reaction, the interaction of enzyme and substrate is considered an equilibrium and the overall reaction as follows: E + S ES E + P

k+1 k-1 k+2 v = velocity, reaction rate KM = Michaelis constant KM = k2 + k-1 k1

C3 - Protein Functions: Signal Transduction

Signal Transduction

• in general the relaying of a signal from one physical form to another
• in biological terms, the process by which a cell responds to signals (can be

intracellular, extracellular). Examples of ‘signals’ (inputs):

• chemicals
• light
• temperature
• electrical (ion gradients)
• other cells (cell-cell contact)
• mechanical sensing

Signal Transduction

Input Output

Generalized Model of Response to Extracellular Signal “Action”

Ligand Receptor Activated Receptor

• Ligand can activate or inactivate receptor
• Output (action) dependent on system and sometime cell type
• In metazoans (multi-cellular eukaryotes), there are about 16 intercellular

classes of signaling systems

“Action”

Ligand Receptor Activated Receptor ~P P~

Example 1: Transmembrane Tyrosine Kinase Receptors

• Ligand binding results in receptor dimerization
• The cytoplasmic (intracellular) domains are tyrosine kinases which

phosphorylate each other on Tyr residue side chains.

• This sets off a series of intracellular events

Ligand Receptor Activated Receptor

Example 2 : Steroid Receptors

nucleus

• The steroid binds to it’s receptor in the cytoplasm.
• The steroid-receptor complex but not the free receptor can move into the

nucleus .

• The steroid-receptor complex binds to specific binding site(s) on the DNA

to regulate gene expression.

Ligand Receptor Activated Receptor

Example 3. Heterotrimeric G-Proteins

GDP GTP GTP GTP GDP

• Ligand binding causes activation of the a subunit which promotes

exchange of GDP for GTP

• In the GTP form, the a subunit and the associated bg subunits dissociate

from the complex.

• Each subunit can go on to initiate a series of intracellular events.

(a b g (a b g complex)

D - Regulation of Protein Activity

Proteins are often regulated such that the ‘activity’ of a protein is not a constant function of its concentration. The concentration of a protein in the cell is a function of the rate of synthesis and the rate of degradation. Both these processes can be regulated.

DNA RNA Protein

Transcription Translation Degradation Synthesis Protein Active Protein Inactive

Regulation of Enzyme Activity A B X

Negative Feedback (Product Inhibition)

A B C D E F X

Mechanistically negative feedback can be by direct competition of the product with the substrate for the active site or it can be indirect through interaction wit the enzyme away from the active site (allosteric).

Regulation of Enzyme Activity A B X

Positive Feedback (Product Inhibition)

A B X

Positive Feedforward

Cooperativity / Allosteric Regulation + +

Hypothetical examples of binding of a ligand to a dimeric protein. The binding curve is very sensitive to the effects on one site on the other. Two independent sites

+ + Cooperativity / Allosteric Regulation + +

Hypothetical examples of binding of a ligand to a dimeric protein. The binding curve is very sensitive to the effects on one site on the other. Two independent sites Positive cooperativity

+ + Cooperativity / Allosteric Regulation + + + +

Hypothetical examples of binding of a ligand to a dimeric protein. The binding curve is very sensitive to the effects on one site on the other. Two independent sites Positive cooperativity Negative cooperativity

+ + Cooperativity / Allosteric Regulation + + + +

Hypothetical examples of binding of a ligand to a dimeric protein. The binding curve is very sensitive to the effects on one site on the other. Two independent sites Positive cooperativity Negative cooperativity

n,1 0.25 0.5 0.75 1 0.01 1 100 10000

Fraction bound vs ligand concentration

+ + Cooperativity / Allosteric Regulation + + + +

Hypothetical examples of binding of a ligand to a dimeric protein. The binding curve is very sensitive to the effects on one site on the other. Two independent sites Positive cooperativity Negative cooperativity

0.25 0.5 0.75 1 0.01 1 100 10000

Positive Cooperativity (n=2, n=3)

+ + Cooperativity / Allosteric Regulation + + + +

Hypothetical examples of binding of a ligand to a dimeric protein. The binding curve is very sensitive to the effects on one site on the other. Two independent sites Positive cooperativity Negative cooperativity

0.25 0.5 0.75 1 0.01 1 100 10000

Negative Cooperativity (n= 0.5)

Allosteric protein: a protein that changes from one conformation to another upon binding a ligand or when it is covalently (chemically) modified. The change in conformation alters the activity of the protein. Historically considered with multi-meric proteins (e.g. hemoglobin).

Allosteric effector (positive)

Ligand

Regulation of Protein Activity by Covalent Modification

The activity of a protein can modified by addition or removal of a chemical group to an amino acid side chain (i.e. - as a substrate for another enzyme). The most common modifications are:

• Methylation (-CH3)
• Phosphorylation (-PO3)
• Nucleotidyl
• Fatty acid
• Myristol

note that many proteins are modified in other ways such as addition of sugar groups (glycosylation) but these are not ‘regulatory’ modifications.

Phosphorylation is the most common mechanism of regulation by covalent modification Kinase - an enzyme that phosphorylates Phosphatase - an enzyme that removes phosphate

Regulation by Localization

Protein activity can be regulated by changing the localization of the protein. This turns out to be a common theme in eukaryotic signal transduction. Localization can be altered allosterically or by covalent modification.

Addition of a fatty acid group can cause a cytoplasmic protein to associate with the cell membrane.

~P P~ ~P P~

Covalent modification of a protein can generate a binding site for another protein.

E - General Considerations

Proteins have a diverse range of functions and a variety of mechanisms

• f regulation. The ability to form networks of proteins acting on

proteins, the sharing of common reaction intermediates and forming multi-step chemical pathways allows for an endless number of possibilities. Some general considerations about protein systems:

• A reaction can behave as a step function (digital, boolean) if there is

significant cooperativity in the system or if there modifying enzyme that works near saturation.

• Since proteins can act in a catalytic manner, there can be signal

amplification.

• Many systems are adaptive, in that the response to signal is not necessarily

constant over time (e.g. a signal transduction system may become desensitized and no loner respond to the presence of a ligand- c.f. heterotrimeric G protein).

EnvZ/OmpR system in E. coli bacteria ~P ~P OmpR EnvZ

Increasing Osmolarity

EnvZ

EnvZ is a histidine kinase (phosphorylates specific histidine residues) in response to changes in osmolarity (salt concentration). The ~P group is transferred to OmpR to form OmpR~P. EnvZ also catalyzes the dephosphorylation of OmpR~P. OmpR~P is a transcriptional regulator of two gene (ompF and ompC). It binds to DNA only in the phosphorylated state.

OmpR~P can activate or repress expression of a gene depending on the position of the binding site relative to the promoter.

~P ~P

X

OFF ON

Activation and repression of the ompF promoter is regulated by a high affinity and a low affinity binding site respectively. Activation of ompC is through a low affinity activator site.

• mpF

+

• mpC

+

Note that OmpR~P is required for both ompF and ompC transcription.

Low osmolarity High osmolarity

• mpF

+

• mpF
• mpC

+ +

• ~P
• mpC

+

~P

ON ON OFF OFF

~P ~P ~P

Osmolarity Protein Level OmpR~P OmpC OmpF

Not an ON/OFF switch but more like a thermostat (i.e. gradients of expression levels).

~P ~P

Regulator Receptor Increasing Signal [Signal] [output signal]

Linear dependence

Playing with Switches

~P ~P

Regulator Receptor Increasing Signal [Signal] [output signal]

Linear dependence Adding Cooperativity

Playing with Switches

~P ~P

Regulator Receptor Increasing Signal [Signal] [output signal]

Linear dependence Adding Cooperativity

Playing with Switches

Adding More Cooperativity

~P ~P

Regulator Receptor Increasing Signal [Signal] [output signal]

Playing with Switches

Approximates a step function (ON/OFF Switch)

Not all pathways will operate in a single cell.

Epidermal Growth Factor Signaling Pathway

http://www.grt.kyushu-u.ac.jp/spad/pathway/egf.html

• Protein interactions
• Protein modification

(Activation/inhibition)

• Protein re-localization
• Transcriptional regulation

Protein Stability: Robust to Site Mutations

Protein function sensitive to changes in active site residues but relative robust with respect to ransom mutations. Tolerance for random mutations in proteins in general ~ 8 - 30% In some studies as much as 25% increased protein stability How does nature select for robustness? Is there a connection between robustness and evolvability?