BC 3001

Bioluminescence

Bioluminescence

The production and emission of light by a living organism.

Chemiluminescence

The emission of light as a result of a chemical reaction.

Aequorea victoria

A species of jellyfish that exhibits bioluminescence.

GFP (Green Fluorescent Protein)

A protein isolated from Aequorea victoria that absorbs blue light (475nm) and emits green light (510nm)

• 238 AA peptide

• Fluoresce originates from Ser - Tyr - Gly Tripeptide

Photophysics

The study of the physical properties and behavior of light.

X-ray crystallography

A technique used to determine the atomic and molecular structure of a crystal.

Tripeptide

A peptide consisting of three amino acids.

Post-translational modification

Chemical modifications that occur to a protein after it has been synthesized.

EGFP (Enhanced Green Fluorescent Protein)

A variant of GFP with improved spectral properties.

FP Spectral Variants

Variants of fluorescent proteins that exhibit different colors and characteristics.

Red Fluorescent Proteins

Proteins isolated from corals that absorb green light and fluoresce red.

FRET (Fluorescence Resonance Energy Transfer)

A process in which energy is transferred between two fluorophores.

Biosensor

A device or construct that detects and measures biological molecules or processes.

Transgenic

Referring to an organism that has had foreign DNA inserted into its genome.

BRAINBOW

A technique used to label neurons with multiple distinct colors to visualize synaptic circuits.

Genetically Encoded FRET Biosensors

Biosensors that utilize genetically encoded fluorescent proteins for FRET measurements.1. Osamu Shimomura:A scientist who discovered aequorin and GFP in 1961.

GFP

Green Fluorescent Protein, a protein that emits green light when exposed to certain wavelengths.

Martin Chalfie

A scientist from Columbia University who studied C. elegans nerve cells using GFP in the 1980s.

Douglas Prasher

A scientist from Woods Hole Oceanographic Institute who sequenced the GFP gene.

E.coli

The first organism in which GFP was used.

Roger Tsien

A scientist from the University of California, San Diego who made modifications to GFP.

Tulle Hazelrigg

A scientist from Columbia University who studied fusion proteins in Drosophila (fruit fly) and preserved the structure and function of both proteins.

Protein Structure

The arrangement of amino acids in a protein molecule.

Enzymes

Proteins that catalyze chemical reactions in living organisms.

Defence

Proteins, such as antibodies, that protect the body against foreign substances.

Structure

Proteins, like collagen, that provide support and shape to cells and tissues.

Storage

Proteins, like ovalbumin, that store nutrients for later use.

Transport

Proteins, like hemoglobin, that carry molecules and ions throughout the body.

Movement

Proteins, like actin and myosin, that enable muscle contraction and movement.

Regulation/Receptors

Proteins, like insulin and insulin receptors, that control and respond to signals in the body.

Amino Acid

The building blocks of proteins.

Hydrophobic

Amino acids with non-polar side chains that repel water.

Polar

Amino acids with polar side chains that attract water.

Acidic

Amino acids with negatively charged side chains.

Basic

Amino acids with positively charged side chains.

Hydropathy Scale

A scale that measures the hydrophobicity or hydrophilicity of amino acids.

Peptide Bond

The bond formed between two amino acids during protein synthesis.

Ramachandran Plot

A plot that shows the allowed regions of dihedral angles for amino acids in proteins.

Structural Motifs

Short segments of protein 3D structure that are spatially close but not necessarily adjacent in the sequence.

Tertiary Structure

The overall 3D structure of a single protein molecule.

Quaternary Structure

The arrangement of multiple protein subunits in a complex.

Post-Translational Modifications

Chemical modifications that occur after protein synthesis.

Phosphorylation

The addition of a phosphate group to a protein, often used for regulation.

Glycosylation

The addition of saccharide groups to a protein, affecting folding, stability, and regulation.

Acetylation

The addition of an acetyl group to a protein, often used for regulation.

Hydroxylation

The addition of a hydroxyl group to a protein, affecting regulation.

Methylation

The addition of a methyl group to a protein, often used for regulation.

Disulfide Bonds

Covalent bonds formed between two cysteine residues, contributing to protein stability.

Ubiquitination

The attachment of ubiquitin molecules to a protein, marking it for degradation.

Polymorphic

Proteins with sequence variability among individuals.

Site-directed Mutagenesis

A technique used to introduce specific mutations into a protein's DNA sequence for studying structure and function.1. Extracellular fluid (ECF):The fluid surrounding cells from which they acquire molecules and ions.

Diffusion

The spontaneous movement of molecules and ions down their concentration gradient.

Active transport

The movement of molecules and ions against their concentration gradient, requiring the expenditure of energy.

Electrochemical gradient/potential

The combination of the chemical gradient (difference in solute concentrations) and the electrical gradient (difference in potential/charge) across a membrane.

Resting potential

The static membrane potential of quiescent cells.

Action potential

The rapid rise and fall of membrane potential in a specific cell location, causing adjacent locations to similarly depolarize.

Voltage-gated ion channels

Channels in the plasma membrane of cells that open and close in response to changes in membrane potential.

Selectivity filter

A segment in voltage-gated ion channels that allows the selective passage of specific ions.

Ion pore domain

A structural component of voltage-gated ion channels that forms the ion-conducting pore.

Voltage sensor

A component of voltage-gated ion channels that detects changes in membrane potential and triggers channel opening or closing.

Tetramerization

The process of four identical subunits coming together to form a functional channel.

Patch clamping

A technique used to record the activity of individual ion channels.

Ion-selective micro-electrodes

Electrodes used to measure the concentration of specific ions.

X-ray crystallography

A method used to determine the three-dimensional structure of molecules, including ion channels.

Bacterial KcsA Channel

An experimental model used to study the structure and function of potassium channels.

Subunit structure

The organization of different segments within a potassium channel, including the selectivity filter, cavity, and internal pore.

Ion selectivity

The ability of a channel to selectively allow the passage of certain ions while excluding others.

Conformational change

A change in the three-dimensional structure of a molecule or protein.

Newton's cradle

A physical demonstration of the conservation of momentum, used here to describe the oscillation of potassium ions within the selectivity filter.

Vestibules

Large openings or chambers in ion channels.

Coordination geometry

The arrangement of atoms and their bonding in a molecule or ion.1. Rod MacKinnon:A scientist known for his work on ion channels.

Ion Channels

Protein structures that allow the flow of ions across cell membranes.

Reason to purify proteins

To provide material for structural or functional studies

Truly accurate method for determination of protein conc.

AA analysis on the hydrolysate

Mostly used for determination of protein conc.

Bovin serum albumin (BSA)

• Low cost, high purity, ready availability

Ultraviolet absorption

Method for determination of protein conc, non-destructive, can be measured continuously, readily available

Lowry method, bicimchoninic method, Bradford method

Used to determine protein conc

Buffer composition (P. Isolation)

Ionic strength (0.1-0.2M)

PH (7-8)

Mostly Tris or phosphate

Anti oxidant

Buffer add

• to prevent inter-/intramolecular disulphide bridges & oxidation

Enzyme inhibitors (buffer)

• Protease inhibitors

Enzyme substrate & cofactors

Buffer add

EDTA

• buffer add

• To remove divalent metal ions

PVP

• buffer add: plant tissue

• To absorb phenolic compounds

Sodium azide

• buffer add

• Bacteriostatic agent

Extrinsic membrane protein isolation

Increase ionic concentration or increase pH(9-12) of buffer

Intrinsic membrane protein isolation

Add detergents (SDS, CTAB, CHAPS)

Mammalian cells

• 10um

• Weak plasma membrane/cytoskeleton

• Easy to disrupt

Plant cells

• 100um

• Fairly rigid cell wall

• Till susceptible to disruption by shear force

Bacterial cells

• 1-4um

• Extremely rigid cell walls

• Gram +/-

Blender Method

Ideal for mammalian / plant tissue

bacteria/yeast with glass beads

Grinding with abrasives

Ideal for bacteria/ plant cells

Presses

Ideal for microbial cells

Enzymatic disruption methods

• Lysozyme: bacteria

• Zymolyase/Yticase: yeast

• Chitinase: fungi

Sonication

Ideal for suspension of cultured cells/ microbial cells

Stability

Denaturation fractionation (heat/pH sensitivity)

Solubility

• salt fractionation: ammonium sulfide (NH4)2SO4

• Organic solvents: ethanol/ acetone/ Butanol

Fine purification

Higher resolution chromatographic methods

Charge

Ion-exchange chromatography, chromafocusing

Affinity

Affinity chromatography (ligands)

Protein sources

• biological fluids (urea,blood plasma)

• Intercellular components

• Recombinant proteins

• Membrane bound components