Protein structure 2

Question 1

Describe the structures

Answer 1

Primary structure – the actual amino acyl sequence.
* Secondary structure – the natural in vivo folding of the primary structure as it falls off of the ribosome (+/- chaperone proteins/inside GroEL/GroES complex) based on the sequence to form 3D structures.
* Tertiary structure – amino acid side-chain interactions to give polypeptide domains in complete folded shape.
* Quaternary structure – multiple polypeptide domains interacting to form a complete protein and/or the binding of biochemical cofactors(FMN, FAD, PQQ, heme b, retinaldehyde etc) and/or metals to the tertiary structure.

Question 2

Tertiary structures

Answer 2

• Formed by other interactions (cf. first year – all except H-bonds which are only secondary) within the polypeptide.
• MANY possible motifs – I’m only going to skim the surface!

Question 3

Predicting helices that span membranes

Answer 3

• Hydrophobicity plots such as the Kyte-Doolittle plot allow us to examine a primary structure for regions that are
  1) hydrophobic enough to form alpha helices
  2) if those helices are long enough to cross a cell membrane. membrane thickness depends on lipid bilayer structure – e.g. Thiobacillus spp. mostly C16:0 and C16:1, in Myxococcus spp., mostly C22:0 to C24:2 – not huge differences in thickness, but they are there.

alpha helices can cluster together to form pores across membranes. Forming tertiary motifs =membrane spanning regions.
You can predict this by looking for helices that are long enough to cross the membrane and hydrophobic enough to stay in a lipid membrane. (this depends on type of membrane and thickness).

membrane thickness in bacteria varies in family level and the thickness is detected by the length of the fatty acids on the lipids.

Question 4

Predicting helices that span membranes

Answer 4

• Aquaporin-1 from Homo sapiensL. contains 7 alpha helices per subunit and 4 subunits are grouped to form a trans-membrane pore which lets water in and out of the cell. Only hole in the middle. sub units can change shape so dictates how open the hole is
• Using the primary structure (provided in a .FASTA) and the
  ProtScale tool, we can run a Kyte-Doolittle plot.
  (it is a hydrophobicity plot)
  above0 =hydrophobic
  below0=hydrophilic
• https://web.expasy.org/protscale/

Question 5

Beta motifs: the beta propeller

Answer 5

• Multiple (usually 4-stranded) beta-meander motifs arranged in the form of ‘blades’.
• Shape varies from a propeller to a shallow bowl.
• Site of protein-protein interaction as quite hydrophobic.
• Active site of many enzymes, in centre of the propeller.

you need a large and small surface area depending on the job.
more like a donut but has a dimple not a hole
prosthetic groups stuck in central region

Question 6

more on Beta motifs: the beta propeller
Where are they most commonly found?
Give an example

Answer 6

• Common in alcohol dehydrogenases, particularly those that deposit electrons onto cytochromes.
• An eight-bladed propeller, which is common in alcohol
  dehydrogenases. Bound Ca2+ ion is shown and the
  bound cofactor PQQ.

(EC 1.1.2.7, MxaFI) from Paracoccus denitrificans

it takes electron from the alcohol and donates them to cytochrome c (respiratory chain) then to terminal oxidase then to oxygen so it can be coupled directly to respiration

CH3OH + 2cyt cL(ox) → HCHO + 2cyt cL(red) + 2H+

Question 7

Beta motifs: the beta barrel

Where are they common in?
Give an example how they work
Give an example of one that is hydrophobic outside and one that is hydrophilic inside

Answer 7

Common in;
1) membrane channels/receptors – barrels can
span a membrane.
2) soluble proteins that have hydrophobic
substrates (as the interior of the barrel is hydrophobic).
They don’t always have to be a full barrel

It is hollow in the middle with gating regions (beta strands) something will bind to make the middle open.
They come in both hydrophobic inside or vice versa
hydrophobic on the outside so that it can stay in the membrane.

How they work
*lets say there is sugar in the environment, this attaches to the outside which then attaches to the outside, something on the inside gets released that will go to the DNA and say there is sugar by knocking another protein off which makes a gene gets expressed for sugar metabolism
They are used as;
*sensors
*channel protein which can be gated or non gated
*signal transduction when in membranes but occasionally used as an anchor to attach it to the membrane (expensive to make to rare)

e.g Crystal structure of the outer membrane active transporter FepA from Escherichia coli

If its hydrophobic inside and hydrophilic outside it is found in the cytoplasm as it dissolves in water, you find these in enzymes that deal with hydrophobic substrates. like alkanes benzene

e.g The Structure Of Ompf Porin In A Tetragonal Crystal Form

Question 8

Alpha solenoid

Where are they found?
What do they do?
Give an example

Answer 8

important in humans
found in kinases and protein phosphatases
found in things that need protein protein interactions and membrane coat proteins so important in tissue recognition in immunology.

In the family of histocompatibility proteins and things that are involved in recognition generally contain alpha solenoids which form very large flexible surfaces for proteins to interact with each other. (could interact with antibodies to recognise self or non-self)

• Found in Eukarya and some Archaea, rare in Bacteria or viruses.
• Stacked pairs of alpha helices (helix-turn-helix motifs) arranged antiparallel, forming a superhelix.
• Flexible.
• Form large, flexible interaction-surfaces for protein-protein interaction: found in enzymes that act on proteins.
• Also found in membrane coat proteins.

it phosphorylates proteins (phosphatases)

Protein Phosphatase 2A bound to TIPRL in homo sapiens

Question 9

Quaternary structures

Answer 9

Formed by polypeptide interactions to hold subunits together by ionic, hydrophobic , disulphide bridges
* Binding of cofactors to proteins – this is often by conserved amino acyl residues so one can predict e.g. ATP-binding sites etc in silico easily.

Question 10

Reminder of information flow

Answer 10

• Operons are groups of genes that
  transcribe as one mRNA strand –this needs in vitro evidence and in silico prediction is not possible.
• Otherwise it is a “gene cluster”

Question 11

Redox co-factors: bound

Answer 11

There are soluble redox co-factors like NADH/etc which are not part of a protein.

The ones that are found in proteins are;
* Found in all redox-active enzymes (EC 1.x.x.x) – including dehydrogenases, oxygenases, oxidases, reductases
* PQQ (pyrroloquinoline quinone) - synthesised elsewhere and bound to the protein
- found in quinoproteins – and TQQ (tryptophan trypto phylloquinone) - formed by post-translational modification of tryptophan residues on the protein itself.
quinones are very hydrophobic and want to stay in the membrane so version of quinone here has something that makes it more hydrophilic so it can be bound to a protein. When protein doesn’t have PQQ is not finished enzyme (can’t do anything)

• Hemes in cytochromes, myoglobin and hemoglobin’s - porphyrin rings centred on Fe(II) —> Fe(III) - variations on a theme heme B and heme C just have different
  side-chains. Flavins (FMN —> FMNH2; FAD —> FADH2) in flavoproteins. Note:
  cytochromes are named for their hemes but formatting is different – cytochrome c contains heme C. Chlorophylls (based on chlorin rings, similar to hemes but with Mg(II) in the centre). Cobalamins and other corrinoids (based on corrin rings again similar but Co(II) in the middle – cf. Vitamin B12) or Ni(II) in cofactor F430

flavo=yellow

Question 12

Bound metals

Answer 12

• Can be ‘naked’ or part of a cofactor.
• Hemes (iron), molybdopterins (molybdenum or tungstun), cofactor F430 (nickel), chlorophylls (magnesium), cobalamins (cobalt) are all examples of metal cofactors
  where the metal is in a ‘frame’.
  Usually made separately and bound to the protein.
• Naked metals found in proteins
  include copper, iron (in Fe-S centres), nickel, manganese, calcium, lanthanum, cerium, zinc, magnesium…

Question 13

Bound metals: examples (find your own!)

Answer 13

• Heme-iron (Fe): cytochrome c, cytochrome b in all Domains, plus hemoglobin, myoglobin in the Eukarya
• “Naked” iron (Fe): soluble methane monooxygenase in the Bacteria, rubredoxins.
• Molybodopterins (actually contain no metal!): nitrogenases (with Mo) and xanthine dehydrogenases (with W) in the Bacteria.
• Tungsten (W): some formate dehydrogenases, acetylene hydratases.
• Nickel (Ni): some hydrogenases.
• Cofactor F430 (Ni): coenzyme-B sulfoethylthiotransferase (EC 2.8.4.1) in the Archaea.
• Chlorophylls (Mg): photosystem proteins.
• Cerium (Ce), lanthanum (La), neodymium (Pr) etc: some methanol dehydrogenases and some RuBisCOs, peptidases.
• Calcium (Ca): some methanol dehydrogenases and some RuBisCOs, calmodulins.
• Zinc (Zn): carbonic anhydrases
• Copper (Cu): cytochrome a, rusticyanins, plasticyanins, azurins, blue copper proteins, particulate methane monooxygenase.
• Cobalamin-cobalt (Co): methionine synthase.
• “Naked” cobalt (Co): some amine oxidases.
• Manganese (Mn): superoxide dismutases.
• Vanadium (V): haloperoxidases, some nitrogenases.

Question 14

Protein Structure and Function II SUMMARY

Answer 14

You should be able to:
* Use a hydrophobicity plot to predict alpha helices that span membranes.
* Recognise an alpha helix pore.
* Recognise a beta propeller and know what they do and why.
* Recognise a beta barrel and know what they do and why.
* Recognise an alpha solenoid and their role etc.
* Recognise a heme group and know what it does.
* Know what the common bound redox cofactors are.
* Know what the common bound metals are

Question 15

How to tell if its a protein or a gene?

Answer 15

If it is written as non-italicised but with a capital letter its is a protein. If it is italicised and first letter is not a capital it is a gene.