Antibodies, molecular motors, + muscle contraction + protein analysis

what are antibodies produced by?

B-cells

____ is the most abundant antibody

IgG

each IgG consists of ____ subunits/ polypeptide chains

4

each IgG consists of ____heavy & light polypeptide chains

2

how are the chains in IgG kept together?

Within immunoglobulin folds, disulphide bonds form a Y shaped protein (cystines)

Disulphide bonds (cysteines) between chains are called _______

interchain

Disulphide bonds within chains are called _______

intrachain

CDR stands for _________________ each containing 3 _______________ (amino acid sequences highly variable) that bind the antigen.

complementarity-determining regions , hypervariable loops

reducing agent used for PAGE (Polyacrylamide Gel Electrophoresis) of IgG

DTT : breaks disulphide bonds so subunits separate

the ______ on an antigen is capable of eliciting an ______ response and can combine with a specific antibody produced by such a response.

epitope, immune,

what is the term describing how IgG can bind to 2 antigens simultaneously?

divalent bonding

what are the 2 reasons antibodies cross link to form a lattice ?

- speeds up removal of antigen
- triggers B-cell proliferation

monoclonal antibodies are a collection of _______ that interact with a ____ antigen site

identical antibodies , single antigen site

what are 3 uses for lab produced monoclonal antibodies?

1. - protein purification and identification in the lab
   

2. identification of disease
   

3. therapeutic agents

Sickle cell anaemia is a genetic disorder caused by a specific ______ mutation in the gene that codes for _____

amino acid mutation, beta-globin

In normal haemoglobin, the 6th amino acid in the beta-globin chain is

glutamic acid (Glu)

in sickle cell anaemia, a single nucleotide change leads to the substitution of glutamic acid with ________

valine (Val)

the substitution of Glu with Val in sickle cell anaemia results in the formation of ________

abnormal haemoglobin called haemoglobin S (HbS).

Misfolded proteins have a tendency to ________

aggregate, meaning they stick together and form clumps.

insoluble clumps formed from misfolded proteins aggregating are called

amyloid (fibrils) eg β amyloid plaques (Alzeimher’s)

what are 3 ways amyloid plaques can disrupt cell function?

• Interfere with cell-to-cell communication and signalling pathways.
  

• Trigger inflammation and immune responses.
  

• Induce oxidative stress, causing damage to cellular components.

The amino acid deletion that results in cystic fibrosis is caused by the loss of ____ at position ____ in the _____ protein

The loss of phenylalanine at position 508 in the CFTR protein (Cystic Fibrosis Transmembrane Conductance Regulator)

Muscle fibres are multinucleated, True or False

True

what are muscle fibres produced by?

cell fusion

________ are microscopic protein filaments that make up muscle cells

Myofibrils

Myofibrils are surrounded by flattened _____ containing Ca2+ called ________________ (nerve impulses cause Ca2+ release)

vesicles, sarcoplasmic reticula

Myofibrils consist of :

• I band contains only thin filaments

• A band contains only thick filaments in the H zone. Darker outer segments contain overlapping thick and thin filaments

The _______ is the contractile unit of muscle

sarcomere

during muscle contraction, does the length of each filament of myofibril change?

Yes -

during muscle contraction, does the length of the sarcomere change?

Yes, it reduces in size due to overlapping/sliding of the filaments

what are the 4 key proteins involved in muscle contraction?

- myosin
- actin
- Tropomyosin

-Troponin

where is myosin found?

in the thick filaments

where is actin found?

thin filaments

where are Tropomyosin & troponin found?

thin filaments

what kind of role do Tropomyosin & troponin have?

inhibitory - they are regulatory proteins in muscle contraction.

The most abundant cytosolic protein in eukaryotes is _____ and its microfilaments are responsible for :

Actin

• Changes in cell shape

• Cell division

• Cellular locomotion

• Endocytosis

• Organelle transport

monomeric form of actin is known as ______ and forms polymers of _______ actin

G-actin, fibrous (F-actin) aka microfilaments

The sliding filament model of muscle contraction describes:

the movement of thick filaments (myosin) relative to thin filaments (actin)

• The myosin head must repeatedly detach and then reattach itself at a new binding site on actin further along the thin filament.

•Ca²⁺ ions from the Sarcoplasmic Reticulum bind to troponin, pulling tropomyosin off the binding sites.

• MH 1st bind weakly to actin forming a cross-bridge, when Pi is released, strong binding occurs & the MH pulls strongly onto actin creating a power stroke

• Contractile force is provided by ATP hydrolysis, which breaks the cross-bridge & resets the MH to their original position, ADP is released (chemical energy -> mechanical energy

Cellular locomotion is mediated by:

treadmilling of actin microfilaments which is driven by ATP hydrolysis

How does Spectroscopy quantify proteins?

- aromatic side chains in amino acid residues absorb UV light

- these then emit light at a new wavelength

4 principle steps in ELISA

1) An antibody against the protein of interest is immobilised on the bottom of the wells.

2) The sample is added to the wells. The antibody binds the protein of interest, and other proteins are washed away.

3) A 2nd antibody (specific for a separate site on the protein of interest) is added to the wells. This antibody has an enzyme is attached. The unbound antibody-enzyme complex is washed away.

4) Binding of the 2nd antibody- enzyme complex is measured by assaying the activity of the enzyme.

*The amount of substrate converted to product indicates the amount of protein present.

proteins are separated in a lab by _____ in a __________ with a ________ gradient

spinning , centrifuge, in a salt gradient

3 steps in the fractionation of proteins:

A salt is added to a solution of macromolecules to a concentration just below the precipitation point of the protein of interest

After centrifugation, the unwanted precipitated proteins are discarded and more salt is added to the supernatant to a concentration sufficient to precipitate the proteins of interest

After a 2nd centrifugation, the desired protein (green) is recovered as a precipitate and the rest is discarded

____________ is an example of a salt that may be used in fractionation of proteins in complex mixtures

ammonium sulphate (NH₄)₂SO₄

precipitation point is the calculated __________ of an ______ in solution

The calculated solubility point of an ion in solution (scale/brine stability calculations).

_______ is the liquid on top of material deposited by _______ or _______

supernatant , settling or centrifugation.

what is the 'salting out' in fractionation of proteins based on?

competition between the added salt molecules and the dissolved solutes

size exclusion chromatography is also known as _________

gel filtration chromatography

what does size exclusion chromatography do?

separates proteins based on their size and shape

In size exclusion chromatography, _____ proteins move through the column more ______ because they can't enter the ____ of the gel, while ______ proteins take _____ as they enter the pores.

larger proteins, quickly , the pores, smaller, longer

procedure of size exclusion chromatography

The protein mixture is applied to a column filled with a porous matrix.

As the solution flows through the column, proteins are separated based on their molecular size

what does Ion Exchange Chromatography do?

Separates ions and polar molecules based on their affinity to the ion exchanger.

2 types of Ion Exchange Chromatography?

- anion-exchange
- cation-exchange

In anion exchange Ion Exchange Chromatography the column resin is _______ charged, attracting _________ charged proteins

positively charged, attracting negatively charged proteins

In cation exchange Ion Exchange Chromatography the column resin is _______ charged, attracting _________ charged proteins

negatively charged, attracting positively charged proteins

principle of Ion Exchange Chromatography:

A charged resin attracts proteins with opposite charges.

what method separates proteins based on their charge?

Ion Exchange Chromatography

what method separates proteins based on their size and shape?

size exclusion chromatography

what method separates proteins based on their binding affinity for to another protein on the column bead?

Affinity Chromatography

what does Affinity Chromatography do?

separates proteins based on their binding affinity for to another protein on the column bead

Affinity chromatography uses a specific interaction between a _____ and a _____ immobilised on a ____

protein and a ligand (eg. antibody enzyme substrate) , matrix

procedure of Affinity Chromatography:

• The mixture is applied to a column with the ligand.

• The protein of interest binds specifically, while others pass through.

• The bound protein is then eluted using a competitive agent.

The most applied affinity system for the purification of antibodies is the _____

Staphylococcal protein A (SPA) and smaller ligands derived thereof

what method separates proteins based on their molecular weight?

Polyacrylamide Gel Electrophoresis

what does the sample buffer for Polyacrylamide Gel Electrophoresis contain?

Sodium dodecyl sulfate (SDS) denatures proteins and confers a negative charge

DTT / β-mercaptoethanol: reducing agents (break disulphide bonds)

what does Sodium dodecyl sulfate (SDS) do in Polyacrylamide Gel Electrophoresis ?

denatures proteins and confers a negative charge

what does DTT / β-mercaptoethanol do in Polyacrylamide Gel Electrophoresis ?

they are reducing agents that break disulphide bonds

principles of Polyacrylamide Gel Electrophoresis

• Samples are loaded into wells in a vertical polyacrylamide gel & a current is passed through the gel
  

• Small proteins migrate quicker through the gel, larger proteins stall towards the top of the gel

what is the isoelectric point sometimes. abbreviated as?

pI

The __________ is the pH at which a particular molecule carries _____ electrical charge

isoelectric point , no net

at a proteins pI it is

immobilised in an electrical field

Isoelectric focusing (IEF)

Form of electrophoresis through a stable pH gradient such that a charged molecule migrates to a position corresponding to its isoelectric point.

______ is a a technique that separates particles according to their ____

Mass Spectrometry , mass

Tandem Mass Spectrometry

Peptides are separated by the first MS and one peptide is sent through a collision cell where it collides with helium atoms

5 steps of X-Ray Crystallography

A. Precipitation of a pure preparation of the protein

B. Crystal formation - solid crystal of pure molecule

C. X-Ray diffraction through the crystal -reveals a pattern

D. Capture of the diffraction pattern and rendering by computer software into mathematical co-ordinates

E. Deposition of the 'Crystal Structure' into the database

Cryogenic-Electron Microscopy (cryo-EM) produces

3D images in atomic detail

Proteins in a sample can be quantified by

U.V. spectrophotometry

proteins can be purified according to their

size, solubility, ionic charge or binding properties

Protein sequence can be determined by

mass spectrometry

Protein structure can be determined by

x-ray crystallography or cryogenic-electron microscopy .