Proteins, structure, function. Posttranslational modifications. Supramolecular assemblies.

Question 1

What functions does a protein have

Answer 1

Biological catalysis - enzymes, inhibitors of enzymes

• Body defence - antibodies and complement of immune system
• Transport, storage move materials around the body – haemoglobin for O2, membrane transporters for glucose, ionic pumps, ferritin for iron, albumin, apoproteins
• Regulatory function hormones, receptors, neurotransmitters, components of signal transduction pathways
• Structural function coverings and support - skin, tendons, hair, nails, bone
• Movement muscles, cilia, flagella
• Maintenance of electrolyte and water balance

Nutrition- source of nitrogen, amino acids and energy

• Diagnostic tool - plasma proteins

Question 2

native protein

Answer 2

• a protein possessing a specific biological function with its defined
  3D arrangement = native conformation

Question 3

conformational change

Answer 3

modification or loss of biological
properties

Question 4

draw a protein structure

Answer 4

Question 6

what is residue
polypeptide
protein

Answer 6

Question 7

what can you divide proteins into

Answer 7

according to composition

• simple proteins
• heteroproteins

according to polypeptides

• monomeric
• oligomeric

Question 8

simple proteins

Answer 8

• only contain amino acid residues
  (e.g., histones, albumins, globulins, keratins, elastins, collagen, …)

Question 9

heteroproteins

Answer 9

• contain other biomolecules (lipids, sugars, nucleotides, phosphate group, metal ions,…)
  = prosthetic groups - covalently or non-covalently bonded to a protein
  (e.g. haemoglobin – haem, flavoproteins – FAD, FMN, immunolgobulin G – saccharides)
• these groups impart additional properties to a protein

Question 10

monomeric

Answer 10

• only a single polypeptide chain is present
• e.g. albumin, trypsin, Mb

Question 11

oligomeric

Answer 11

• two or more polypeptide chains = subunits are present
• the subunits are typically held together with non-covalent bonds
• subunits – same/distinct
• e.g. Hb, key enzymes of metabolism, G-proteins

Question 12

what shapes can a protein form

Answer 12

• fibrous
  -globular

Question 13

fibrous protein info

Answer 13

Form long fibers and mostly consist of repeated
sequences of amino acids – a single type of secondary structure
* Insoluble in water, chemically stable
* Structural and supporting function
form used by connective tissues
* Silk, collagen, elastin, -keratin, myosin

Question 14

globular protein structure

Answer 14

• Tend to form ball-like structures – several types
  of secondary structure
• Soluble in water, sensitive to changes of
  physicochemical conditions – pH, temperature,
  salt concentration
• Most proteins of cells and extracellular fluids
• Albumin, myoglobin, haemoglobin, enzymes, Ig

Question 15

4 levels of protein structure

Answer 15

primary
- actual sequence
secondary
- alpha helix , beta sheet , bends and loops
tertiary
- folded polypeptide chain due to interactions of amino acid chains
quaternary
-association of 2 or more polypeptide chain to form molecule

Question 16

primary structure in more depth

Answer 16

• Primary structure = sequence of AA in polypeptide chain and position of disulfide bonds

Common base - all proteins have the same covalent backbone (polymer of AA)

Difference - order and presence of individual AA → side chains
The AA sequence determines the spatial arrangement of a protein molecule. it can predict mechanism of a protein action (enzymes)

it could also lead to abnormal function
→ disease e.g. Hb → HbS → sickle cell disease
collagen → fragility and bending of bones
(Osteogenesis imperfecta)

Question 17

posttranslational modification of AA in the polypeptide chain

Answer 17

• Additional modification of AA residues in proteins (some co-translational) can happen.
• Performed specifically by enzymes
• Modification of protein properties, stabilization, participation in the regulation of their
  function (activity), etc.

some examples

Acetylation of terminal -NH2 (Lys)
- Reduces protein basicity → reduces ionic interactions

Methylation of terminal -NH2 (Lys, Arg)
- Increases protein basicity → enhances ionic interactions

Acylation (myristoyl, palmitoyl), prenylation (farnesyl) of terminal AA
- Helps in the anchoring of proteins in the membrane

Glycosylation
- Ser, Thr - O-glycosylation, Asn - N-glycosylation
- Mainly extracellular proteins → increase in solubility, stabilization of conformation, receptor recognition and resistance to proteolysis

Hydroxylation of Pro, Lys
- increase stability - typical for collagen fibers

• Cleavage or excision of part of a polypeptide chain
• Importance in protein activation
  E.g. proenzyme → enzyme,
  fibrinogen → fibrin,
  proinsulin → insulin
• Phosphorylation - OH - Ser, Tyr, Thr
• Reversibly changes activity (enzymes) or affinity
• Mediated by kinases (phosphorylation)
  and phosphatases (dephosphorylation)
• Ubiquitination
• Attachment of a small protein ubiquitin via NH2 lysine
• Important for protein stability and degradation → serves
  as “molecular clock” - determines the age of a protein
  (signalises that the protein should be degraded by
  the proteasome)

Question 18

more info on secondary structure

Answer 18

the aa residues cause the chain to fold and the structure also results from the properties of peptide bonds.
stabilised by hydrogen bonds and disulfide bridges .

Question 19

alpha helix and info

Answer 19

Question 20

b pleated sheet

Answer 20

Question 21

bends and loops

Answer 21

• They tie together different segments of a polypeptide chain
• β-Bends
  (reverse turns, β-turns)
• its short segments - made up of 4 AA residues
• reverses the direction of the main polypeptide chain (compact shape of molecule)
• connect regions of more regular secondary structure (a-helix, b-sheet)
• stabilized by the formation of hydrogen and ionic bonds
• its less common, but up to 1/3 of AA in globular proteins
• Random coil
• region of a polypeptide chain without regular repeating

Question 22

more info on tertiary structure

Answer 22

• it is the Spatial arrangement of the polypeptide chain (with its secondary structure) due to
  interactions between SIDE CHAINS of AA residues
• Is pre-determined by the primary structure and its formation is mostly spontaneous
• In a linear sequence, very distant AA residues interact together → leads to a stable,
  compact spatial arrangement of the protein molecule

Question 23

what is a domain in a tertiary structure

Answer 23

Domain = a basic independent functional and structural unit of proteins with its tertiary structure (-50-350AA)
- usually compact globular regions separated by
a disorganized section

• most proteins have two or more domains
• each domain has its biological function
  e.g. ligand binding,
  DNA binding,
  binding to another protein,
  contains a catalytic site, …

Question 24

quaternary structure

Answer 24

Only in some proteins → composed from two or more subunits
(a subunit = a polypeptide chain) → oligomers go to polymers

• Held together by non-covalent interactions
• Subunits – same or different → subunits may either function independently
  of each other or may work cooperatively
• Quaternary structure typical for proteins
  whose functions in the body are regulated
  e.g. Hb, key metabolic enzymes, G-proteins,…

hemoglobin and apoferritin

Question 25

protein folding

Answer 25

* A multi-step process . mostly spontaneous - protein captures the structure with the lowest energy level ( it picks one with low energy , gives it more and then when the protein is folded it has less energy then it started with ) * The arrangement is not random, there is only one final biologically active conformation which is defined by the primary structure * The driving force is the hydrophobic character of some AA residues and the formation of hydrogen bridges between hydrophilic AA residues ( hydrophobic interaction , hydrogen bridges cause the responsible mainly for structure )

Question 26

protein folding which is not spontaneous

Answer 26

Not all proteins form their native conformation spontaneously → some require * Molecular chaperones - which are proteins that assist in the folding process - major classes: -heat-shock proteins (HSP-70, HSP-90) - chaperonins (HSP-60) * Functions: - Bind reversibly to unfolded polypeptide segments, assist to achieve active 3D structure → prevent misfolding and premature aggregation - Block the folding of certain proteins that must remain unfolded until they have been translocated across membrane - Are able to reconstruct denatured forms of polypeptides - Assist in protein destruction, if a protein cannot be folded into its proper conformation so its very necessary , deficiency in it can lead to certain diseases and protein misfolding can lead to the targeting of inappropriate cellular location - to a protein degradation, (loss of function and deficiency) - to aggregation and resistance to proteolysis (formation of protein deposits in cells)

Question 27

changes in protein conformation

Answer 27

* Denaturation - Destruction of a native conformation = (quaternary), tertiary and secondary structure by changing the physico-chemical properties of the environment - Mostly irreversible, accompanied by coagulation (= the disorganized protein strands will clump together) - Causes: → change in physical properties (e.g. coagulation of protein chains - solidification of egg white at elevated temperature) → loss of biological activity (e.g. loss of resistance to degradation - the reason for the presence of HCl in the stomach) * Allostery = allosteric effect - Alteration to protein conformation induced by ligand (allosteric modulator) binding to allosteric site - Only in allosteric proteins = 2 or more subunits (= with quarternary structure) - Ligand = ion (H+), small molecule (ATP, cAMP), protein (calmodulin) - Ligand binding leads to a change in conformation = modification (regulation) biological functions - activation/deactivation (e.g. regulation of affinity Hb - O2 , enzyme - substrate)

Question 28

is reversible denaturation of proteins possible

Answer 28

* For some proteins, denaturation may be reversible * E.g. denatured ribonuclease

Question 29

effect of ph on shape of proteins

Answer 29

Changing pH will alter charge on protein ↓ This alters their solubility and may change their shape and biological properties

Question 30

protein folding diseases

Answer 30

protein conformational diseases - diseases caused by pathological conformation of proteins * Causes: - changes in the primary structure of proteins, defects in chaperones. * Consequences: - loss of function (lack of functional protein) - accumulation and formation of insoluble aggregates (tissue damage) * Types of protein folding diseases: - Familial → the disorder is genetically inherited and symptoms appear during childhood - Sporadic → primarily due to aging or to an incorrect lifestyle (not associated with gene mutations;patternless and characterized by a late onset - Transmissible → mainly prion-associated diseases

Question 31

examples of protein folding diseases

Answer 31

Question 32

amyloids

Answer 32

* Pathological polymeric forms of polypeptides / proteins → fibrils, plaques * High proportion of β-pleated sheet (β-structure) → so its insoluble, resistant to degradation by proteinases → accumulation of it inside/outside cells → damage * Origin from normal cellular proteins (soluble and degradable) → 2 possiblities of formation: 1) Naturally contain β-structure and under certain conditions they start to form aggregates 2) Transformation of secondary structure is induced → β-structure formation →intra-/intermolecular interactions → aggregates formation * Mutation significantly accelerates amyloid formation * Approximately 27 proteins capable of forming amyloid have been described and cause disease * Sickle cell anaemia - HbS = hemoglobin mutation (Val – Glu) →polymer formation * Alzheimer’s disease -amyloid –β - aggregation and accumulation * Parkinson’s disease - α-synuclein - aggregation and accumulation so amyloids generally cause disease

Question 33

prions

Answer 33

* Prions = Proteinaceous infectious only - they are proteins which are capable to transfer diseases among organisms (without participation of DNA, RNA) * All known prion diseases in mammals affect the structure of the brain or other neural tissue * Types of prion proteins (PrP): - normal (physiologic) = PrP, PrPc or PrP-sen – constituent of brain tissue of all mammals - abnormal (pathologic) = PrPsc or PrP-re

Question 34

give some examples of supramolecular structures

Answer 34

* Large aggregates of proteins - subunits of the same or of different types → fibres, tubes, spheroids, plaques * Aggregation determines biological function – (actin, tubulin), collagen, protein shell (capsid) of viruses, actin-myosin complex, chaperonins, proteasome Examples of protein aggregates

Question 35

plasma proteins

Answer 35

* Plasma - has hundreds of different proteins - different size, concentration, function * Function of plasma proteins: - Nutritive - Colloid osmotic pressure - Transport of minerals, hormones, vitamins, drugs, - Buffering action (stabilisation of blood pH) - Immunological function (body defence) - Role in blood coagulation and fibrinolysis - Enzymes and inhibitors of enzymes - Viscosity of blood * Production of plasma proteins: - Liver – majority - B-lymphocytes – (immunoglobulins, enterocytes - apoprotein B-48) * Concentration of plasma proteins: 65-85 g l-1 (mostly in serum) * Lower concentration can lead to hypoproteinaemia – malnutrition, malabsorption, liver diseases, nephrotic syndrome, etc. * Higher concentration – can lead to hyperproteinaemia – dehydration, some chronic diseases For diagnostic purposes the levels are evaluated: -Total protein concentration and albumin concentration (concentration of globulins = c total- c albumin

Question 36

separation of plasma proteins

Answer 36

According to the movement in electric field: - by electrophoresis - cellulose acetate sheets (more frequent) or agarose gel ( i think you have done this ) - proteins are separated into 5-6 fractions: albumin, globulins , alpha 1 ,2 and beta 1,2