Protein fundamentals 2 PDF

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L3: Protein Fundamentals 2- Understand the meaning of primary, secondary and tertiary and quaternary structure of proteins - Understand secondary structure elements and how these are stabilised - Structure and properties of peptides - Understand the Ionization behaviour of amino acids and peptides o...

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L3: Protein Fundamentals 2 -

Understand the meaning of primary, secondary and tertiary and quaternary structure of proteins Understand secondary structure elements and how these are stabilised Structure and properties of peptides Understand the Ionization behaviour of amino acids and peptides o Characteristic structure and naming of secondary structure o The folded form of proteins and types of proteins o Basic classification of proteins and peptides based on their biological activities and size

Secondary Structure elements in peptides and proteins These consist of sheets and helices (alpha helices and beta sheets):

Beta sheets can be parallel or antiparallel:

Alpha helices have compact packing and are stabilised by H-bonding, dipole moments and stacking of different R groups on amino acids:

Characterisation of secondary structures: These are shown in Ramachandran Plots as below: (plotting the angles) -

Alpha is noticed at the bottom in a cluster Beta is shown at the top Side red is proline which has a characteristic psi angle

L5: Protein Fundamentals 3 -

The most common macromolecular drug targets

Macromolecules as drug targets / receptors: Receptors specifically recognise and bind to a compound, and acts as a physiological signal transducer / mediator of effect. It must be specific and have an effect, with a specific location. Most drug targets are lipids, carbohydrates, nucleic acids and proteins. Drug / lipid interaction E.g. Amphotericin B (IV antifungal) Structurally, it has two sides – one hydrophilic (OH’s) and one hydrophobic. This packs in a way where the hydrophilic part faces inwards, and hydrophobic region faces cholesterol and lipids on the outer area. This forms a hole in the cell membrane, which then kills the cell!

Drug / carbohydrate interaction Carbohydrates represent the most abundant class of molecules in nature (these are all present outside a cell). All cell surfaces are coated with complex carbohydrates that act as recognition molecules for other cells, functional molecules and pathogens. These are involved in indicating disease more than interacting w drugs. -

E.g. antibodies and carbohydrates Glycomimetics interact with carbohydrates in treatment of influenza

Drug / nucleic acid interaction

DNA has major and minor grooves: -

Major – nitrogen and oxygen of base pairs pointing INWARDS Minor – N and O point outwards

Major groove is more dependent on base composition and is easier to target by drugs that distinguish DNA (may be the site for protein recognition of specific DNA sequences or regions) They can stack and bind into DNA essentially.

Drug / protein interaction Drugs bind in deep pockets within interior of protein. Binding on surface is also possible but may prevent protein-protein interactions.

TYPES OF RECEPTORS: 4 MAIN

Drug / enzyme interaction

E.g. Acetylcholine esterase binds to the receptor that cleaves acetyl and choline. Alzheimer’s disease occurs when acetylcholine is broken down in excess, and by binding to the enzyme, the drug inhibits its action (breaking down of acetylcholine).

Regulating concentration of Ion concentration in cells – ION CHANNELS Gated ion channels – regulate transport of ions across cell membranes and play a role in nervous system.

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Proteins and protein structure Protein function

1st structure – amino acid sequence 2nd structure (alpha and beta) 3rd structure – 3D arrangement (folding) 4th structure – arrangement of 2 or more 3D chains

Membranes – complex lipid-based (and protein) structures that form pliable sheets. These act to separate a cell from its surroundings. These are around 3-10nm thick, with two leaflets of lipids (bilayer – this is asymmetric as some lipids are preferably inside whereas some are outside).

They are formed spontaneously in (aq) and are stabilised by non-covalent forces such as the hydrophobic effect. Proteins can span lipid bilayer (embedded within bilayer). Eukaryotic cells have various internal membranes that divide the internal space into compartments.

These membranes contain two extreme states of bilayers: Temp-dependent: -

Ordered with a cooled temperature (stiff) Disordered with increase in heat (high temp)

Receptors in membranes -

Neurotransmitter (e.g. acetylcholine receptor) Pheromones (taste and smell receptors)

Channels, gates and pumps -

Nutrients ions

Enzymes -

ATP synthesis

These all require a membrane to function as they are embedded within it.

Sterols and hopanols in membranes – these increase membrane rigidity and permeability Primarily cholesterol in animals, phytosterols in plants, ergosterols in fungi.

Long-chain fatty acids – lipoproteins (covalently linked lipid to protein) that are embedded into a cell membrane. The lipid segment can become part of the membrane with the rest of the protein ‘anchored’ to the cell. This allows for protein targeting (‘farnelysation’ - Proteins can be targeted to the inner leaflet of the plasma membrane).

If a drug needs to transport inside a cell, we need transport across membranes. -

Passive diffusion of polar molecules involves desolvation and has a high activation barrier (a) it then gets resolvated once leaving the membrane

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Transport across membrane via proteins is an alternative (b) o Permeases / transporter proteins o Symport (requires two molecules to pass the transporter at once) o Antiporters (take one in and out of membrane at the same time) o Uniport (transports a single molecule)

We can drive ATP chemically to transport protons: energy from proton gradient can be used to synthesise ATP  used for active transport

We can use drugs to interfere with signalling processes by targeting receptors that lie on the synapse.

Macromolecules as drug targets -

Ligand gated ion channels e.g. Ca2+ (excitatory – allows flux of positive ions) torpedo nAChR

L6: Protein Fundamentals 4 Methods to characterize and separate peptides and proteins We purify proteins in order to study it, understand its structure and function, use it in biotechnology and medicine. To do this, we rely on physical properties and how they are different from other molecules/other proteins to get pure proteins. What do we purify proteins from? -

Purify from tissues (homogenise) Cells (break open cells – detergents, enzymes etc) Remove insoluble materials (centrifuge) Complex solutions Other excess material

Properties of proteins: Proteins are made of strings of amino acids and consist of a common polypeptide backbone. The specific sequence of amino acids defines the 3D fold of a protein and its unique function.

Size  how many amino acids in polypeptide chain, how many chains? Only the size of a chain won’t tell the relative sizes of proteins. Gel electrophoresis, gel filtration/size exclusion chromatography

Shape  globular (sphere-like, like the red one on the right) or elongated (fibrous ish) Ion exchange chromatography, electrophoresis

Charge (+- and how much)

Solubility  hydrophobic/polar, insoluble/soluble in (aq) (e.g. myoglobin has a lot of charge distributed outside of the protein, making it quite soluble. However there are almost equal + and – properties, making it overall soluble. Salting in/out, detergents

Binding  does it bind to a particular target? This can somewhat be determined from the amino acid sequence Affinity chromatography Column Chromatography

Separation by size  this only works when materials to be separated are significantly different in size

Separation by charge  pI of a protein = pH when net charge of protein is zero (zwitterions or equal balance of charge) pH < pI  protein has more protons = + charge pH > pI  less protons = - charge Manipulating pH of a protein allows us to change it in relation to other species we want to purify it from.

Ion-exchange chromatography  can exchange anions or cations. The resin must have the opposite charge to the protein so they bind under low salt concentrations (salt is ionic so it may interfere). We can then wash away proteins of the opposite charge. Then elute under high salt concentrations (higher than desired protein conc). Therefore the protein that was originally binded to resin will detach and separate due to being replaced by the salt.

Separation by Affinity  taking advantage of a protein’s natural affinity (e.g. antibodies bind particularly to one protein but not the other) or develop an affinity (engineer protein tags that will specifically have an affinity to that protein – usually recombinant). 1. Add ligand to solution that causes this interaction between the protein and receptor. These will bind as everything else is washed out 2. Add another solution that inhibits the linkage between the protein and receptor to take out the particular protein E.g. His-tag.

These methods can be used in conjunction to make the protein as purified as possible.

Analysing proteins  Electrophoresis -

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An electric field pulls proteins according to their charge within a gel matrix (hinders mobility of protein according to size and shape) o In this method, smaller molecules will move faster as opposed to chromatography SDS (detergent – these lyse cell membranes) binds to and unfolds proteins o Coats proteins in a uniform negative charge o Can be used to calculate MM of a protein o Can be used to determine purity of sample

To identify a sample protein: Edman’s Degradation -

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‘walk through’ a peptide sequence by adding phenyl-isothiocyanate (high pH) to bind to N-terminus and remove segment as residue. This identifies that segment and purifies the remaining peptide through repeating this segment Can only work on small sequences Very tedious

Figure 1: protein purity test via gel electrophoresis

Modern -

DNA sequencing to infer protein sequencing and using mass spectrometry for MM and sequence protein (in fragments) Faster, cheaper

Protein concentration  Colourimetry For complex mixes of proteins or low absorbance proteins. Creating a standard curve to workout concentration of unknown. Aromatic amino acids  spectroscopy UV-Vis: aromatic amino acids / proteins absorb light in UV region (275-280nm) using beer law (A=ecl)...