Structure and function of Proteins - created from Mind Map

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<ul><li>proteins are used to build and repaiir tissues, they are made from polypeptides and involved in every celluilat process as they are the most abundant macromolecules in animals </li></ul>

fibrous<br><br><ul><li>fibruos proteins are stuctureal and insoluble. protein with an elongated shape. Fibrous proteins provide structural support for cells and tissues. There are special types of helices present in two fibrous proteins α-keratin and collagen. These proteins form long fibers that serve a structural role in the human body.</li></ul>

globular<br><br><ul><li>globular proteins are functional and soluble. Globular proteins proteins are one of the common protein types (the others being fibrous, disordered and membrane proteins).Globular proteins are somewhat water-soluble (forming colloids in water), unlike the fibrous or membrane proteins.</li></ul>

Protein sturcture

sequence--> stucture--> function

functions of...

does size matter?<br><br><ul><li>proteins range in size from small proteins like insulin to large proteins like glutamate dehydrogenase, which has about 40 separately folded chains, the bigger the protein, and specifically upon the exceeding of proteins upwards from 50,000 as MWm it is found they are comprised of 2 or more folded chains and into dimers and trimers. </li></ul>

amino acids<br><br><ul><li>There is genarally an asymmetric alpha-carbon in the centre and a side chain which is hydrogen in the simplest case of glycine. this expands to a methyl group for alanine etc. there is a charged carboxyl and amino groups on the elft and top of the alpha carbon</li><li>IF amino acids are found in excess if can be detrimental to health</li></ul>

dislide bonds<br><br><ul><li>Disulfide bonds in proteins are formed between the thiol groups of cysteine residues by the process of oxidative folding. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds.</li><li>insulin has 2 chains and 3 disulfide bonds </li></ul>

permanent wave<br><br><ul><li>

Extensive cysteine cross-linking occurs in many

fibrous proteins including hair (depicted here), and showing the “permanent

wave” curling effect that is achieved by breaking and reforming different

disulfide bonds in the amino acid chains in hair strands.

</li></ul>

silk<br><br><ul><li>

The unusually smooth beta-sheet structure in silk

protein (fibroin), which has a very even and smooth surface due to the high and

repetitive content of mostly two amino acids, glycine and alanine that make up

approx. 75% of the protein.

</li><li>

Dragline silk has a tensile strength of 200,000 psi - stronger than

steel and similar to kevlar - and it is flexible as well.

</li><li>

b-sheets

with disordered helices and b-bends:

Graphite / fibreglass tennis racquets use the same principle for

strength and flexibility

</li></ul>

colagen<br><br><ul><li>

Collagen makes up about 33% of all protein in the

human body, and is present in skin, bone, connective tissue (tendons,

cartilage, ligaments, the cornea of the eye). It is extremely strong by virtue

of its triple helical structure that forms cable-like and very strong fibres.

</li></ul>

biding poccket<br><br><ul><li>

The binding pocket of one of four haem

(porphyrin) groups at the centre of the four

subunits in haemoglobin. The Fe iron is held in the centre of

the haem ring and is able to form a covalent

attachment to molecular oxygen, which gives the protein its oxygen carrier

function. Various nearly amino acid side groups complete the geometry of the

binding pocket and enable the approach and binding of substrate (oxygen) and

protein (haemoglobin). Note that a histidine ring N from the

F8 histidine (bottom) helps to hold Fe covalently within the four pyrrole ringed porphyrin plane which also covalently coordinates

the Fe binding. One of the 6 Fe unsharde electrons is left to bind molecular

oxygen. This is the way that oxygen is “dissolved” from the air into solution

(captured by haempglobin) and delivered to the tissues.

</li></ul>

carbon monoxide poissoning<br><br><ul><li>

An interesting comparison of the coordination chemistry of competitive

binding of molecular oxygen and carbon monoxide to the Fe atom in haemoglobin. Note that the very large difference in

affinities (CO ~ 200 times higher binding affinity than O2)

is due purely to the geometry of binding (O2 binds in an end-on-end bent arrangement,

whereas CO binds in a direct line. This directness enables CO to be bound much

more readily than the angular O2-Fe complex.

</li></ul>

enzymes<br><br><ul><li>

A simplistic view of how enzymes work in an aqueous

environment. Water surrounds the enzyme (blue cube) and substrate (red star

burst) and most of this water must be “expelled” from the region between the

approaching molecules. This expulsion of water (equivalent to the Activation

Energy of substrate-enzyme binding) is an energetically favorable and

spontaneous process made possible by the high delta G negative free energy

value imparted by the increase in entropy (disorder) in the system as the water

is dispersed from the ordered shells around the substrate and enzyme.

</li></ul>

ABC transporters<br><br><ul><li>

PROKARYOTES:

Uptake of nutrients (ions, sugars, amino acids, vitamins, peptides);

sporulation; pathogenesis; translation; DNA repair; multidrug resistance;

export of lipids and capsular polysaccharides; cell division; plant nodulation;

osmotic homeostasis.

EUKARYOTES:

Multidrug resistance to cytotoxic drugs; inherited diseases; efflux of dietary

toxins; antigen processing; cholesterol and lipid trafficking; vision; insulin

release.

covers all species: </li></ul>

primary<br><br><ul><li>sequence of amino acids in polypeptide and are stabilised by peptide bonds</li></ul>

secondary<br><br><ul><li>formation of alpha helecies and beta pleated sheets in polypeptide stabilised by hydrogen bonding between groups along peptide backbone </li></ul>

tertiary<br><br><ul><li>overall 3D shape of polypepride this is stabilised by bonds and interactions between R groups or between R groups and peptide bonded backbone </li></ul>

quaternary<br><br><ul><li>shape produced by combinations of polypeptides and this si tabilised by other interactions and bonds and between peptide backbones of different poly peptides </li></ul>

enzymes<br><br><ul><li>biological catalysts, lower activation energy required for regulation of homeostasis </li><li>e.g. trypsin, poteasem polymerase </li></ul>

storage<br><br><ul><li>Storage proteins are biological reserves of metal ions and amino acids, used by organisms. They are found in plant seeds, egg whites, and milk. Ferritin is an example of a storage protein that stores iron. Iron is a component of heme, which is contained in the transport protein hemoglobin and in cytochromes.</li><li>e.g. casein, ovalbumin </li></ul>

transport<br><br><ul><li>Carrier proteins are proteins involved in the movement of ions, small molecules, or macromolecules, such as another protein, across a biological membrane. Carrierproteins are integral membrane proteins; that is, they exist within and span the membrane across which they transport substances.</li><li>e.g. heamoglobin, serum albumin </li></ul>

contractile<br><br><ul><li>Contractile proteins are proteins that mediate sliding of contractile fibres (contraction) of a cell&#x27;s cytoskeleton, and of cardiac and skeletal muscle.</li><li>e.g. myosin and actin </li></ul>

protection<br><br><ul><li>Proteins on an animal&#x27;s cells that either a) brush off a complement molecule as it starts to bond to the cell membrane, or b) decompose any complement which does bond to the membrane. The proteins protect us against the attack of our own complement system.</li><li>antibodies and fribrinogen</li></ul>

toxins<br><br><ul><li>A poisonous substance, especially a protein, that is produced by living cells or organisms and is capable of causing disease when introduced into the body tissues but is often also capable of inducing neutralizing antibodies or antitoxins.</li><li>e.g. botulism toxin. snake venom</li></ul>

hormones<br><br><ul><li>Hormone: A chemical substance produced in the body that controls and regulates the activity of certain cells or organs. Many hormones are secreted by special glands, such as thyroid hormone produced by the thyroid gland. Hormones are essential for every activity of life, including the processes of digestion, metabolism, growth, reproduction, and mood control. Many hormones, such as neurotransmitters, are active in more than one physical process.</li><li>insulin, oestrogen, growth hormone</li></ul>

structure<br><br><ul><li>Structural proteins are fibrous proteins. The most familiar of the fibrous proteins are probably the keratins, which form the protective covering of all land vertebrates: skin, fur, hair, wool, claws, nails, hooves, horns, scales, beaks and feathers.</li><li>e.g.

cells, hair, viral coats, feathers, collagen,

skin, bone, cartilage, blood

vessels

</li></ul>

polar<br><br><ul><li>

Serine

Threonine

Asparagine

Glutamine

Aspartic acid

Glutamic acid

Tyrosine

Arginine

Histidine

Lysine

Cysteine

</li></ul>

non-polar<br><br><ul><li>

Glycine

Proline

Alanine

Isoleucine

Leucine

Valine

Tryptophan

Phenylalanine

Methionine

</li></ul>

peptide bond configuration<br><br><ul><li>

Two amino acids condensed into a

dipeptide with the peptide bond shown in red between the carboxyl group of the

first amino acid and the amino group of the second amino acid. Water is eliminated as the peptide bond forms

(OH from the carboxyl and hydrogen from the amino group). The peptide bond is a

partial double bond due to the delocalisation of the electron pair from the C=O across

the bond to the N-H group. Note the central alpha-carbon of each amino acid

remains intact with 4 differnet groups attached and the side chain shown

in green and always found in ‘trans’ (opposite sides of the chain). The right

figure shows the disallowed ‘cis’ configuration with the sides groups on the

same side of the chain and steric hindrance (orange semi circles) preventing

stability of this type of configuration.

</li><li>

Alpha-carbons (arrows) either side of a peptide bond (red) - between

two amino acids - are in the trans configuration.

</li></ul>

free rotation<br><br><ul><li>

A pentapeptide of 5 amino acids with 4 dipeptide bonds

(the ones in the centre of each bluish plane with the C=O and N-H shown at

either end of the rigid bond. Note that free rotation is possible about either

end of the alpha-carbon.

</li></ul>

secondary structure

helix<br><br><ul><li>The alpha-helix has vertical H-bonds between

peptide bond C==O and N-H</li><li>The alpha helix (α-helix) is a common secondary structure of proteins and is a righthand-coiled or spiral conformation (helix) in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier ( i + 4 → i {\displaystyle i+4\rightarrow i} hydrogen bonding).</li></ul>

sheet<br><br><ul><li>Beta-strands

are almost always arranged into sheets as shown here with interchain H-bonding</li><li>Beta sheets consist of beta strands (also β-strand) connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A β-strand is a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an extended conformation.</li></ul>

mixed

alpha helix

Beta sheet

covers all species

diseases associated

multidrug resistance<br><br><ul><li>a barrier to chemotherapy, in australia cancer attributes to 28% f all deaths and MDR occurs in 50% of patients and results from over-expression of P-glycoprotein </li><li>

Three ABC transporters, namely, ABCB1

(P-glycoprotein), ABCG2, and MRP1, are known to

elicit multidrug resistance when overexpressed in situations where cancers are

challenged with one or more cytotoxic drugs. The drugs de-repress the promoter

and allow overexpression in cancer cells, resulting in massive efflux (export)

of the cytotoxic drugs, thereby rendering the anticancer chemotherapy

ineffective. The major MDR ABC protein of this group of three is ABCB1

(P-glycoprotein); although ABCG2 is implicated in breast cancer especially.

</li></ul>

conserved architecture<br><br><ul><li>

The conserved architecture of ABC

transporters, with two transmembrane domains and

two cytosolic ATP-binding cassettes. This architecture shows the greatest

divergence in the TMD sequences, both in the number of TM helices (4 to 10 for

each half) and the amino acid sequences. The NBDs are highly conserved (see

slides further on). Note that though this slide shows a single polypeptide, ABC

transporters are the evolutionary result of fused, and originally identical

genes. There are three types of configuration, involving single polypeptides,

half transporters (one each TMD and NBD), and four separate subunit

arrangements (four separate TMDs and NBDs) still arranged as above as

non-covalent oligomers).

</li></ul>

alignment of ABC ATP-Binding cassettes<br><br><ul><li>

An amino acid primary sequence alignment of several

ATP-binding cassettes to indicate the highly conserved sequences. The same

degree of similarity occurs across the whole ABC superfamily. The identical

amino acid single letter code (vertically) shows the highly conserved sequence

(grey and coloured

regions). The three highlighted coloured motifs are especially conserved in ABC

transporters. The Walker A &amp;amp; B motifs are found in all proteins/enzymes

that bind ATP and Mg, but only ABC transporters have the unique “signature” C

motif sequence.

</li></ul>

MEchanism of ATP hydrolysis in an ABC transporter

histidine permease ATP-binding cassette dimer<br><br><ul><li>

The first ATP-binding cassette dimer

X-ray crystal structure published in Nature in

1998; shown here in side and top views. Despite the long awaited appearance of this first

structure, we at UTS (Dr

George’s group) were suspicious of the integrity of this structure. For

instance, it does not seem to conform to the Gibb’s equation for spontaneous

free energy and entropy. The assembly of proteins into 3-D configurations

should be spontaneous and generate a large increase in entropy by the release

of the ordered surrounding water shell back to the bulk solvent. This structure

looks too open (see slides further on).

</li><li>clue: highly conserved A, B, and C motif

sequences among the residual and also highly conserved ATP-binding cassette.

Most of the grey conserved sequences make up the structural

scaffold that allows the cassette NBD to fold in

such a way to create the stable geometry around the ATP catalytic site. The

site itself is even more highly conserved in having to bind ATP, hence the A

and B motifs. But what of the ‘C’ motif and the Nature paper HisP dimer? </li></ul>

p-glycogoprotein<br><br><ul><li>P-glycoprotein 1 (permeability glycoprotein, abbreviated as P-gp or Pgp) also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1) or cluster of differentiation 243 (CD243) is an important protein of the cell membrane that pumps many foreign substances out of cells.</li><li>

more

than 400 substrates and moderators of ABCB1. Such a vast spectrum of substrates

(including all cytotoxic drugs) gives ABCB1 the dubious distinction of having

the widest spectrum of any multidrug transporter.

</li></ul>

how do subunits fit together?<br><br><ul><li>

The second

“nail” in the HisP open

dimer “coffin” is that proteins fold and associate as dimers, trimers,

tetramers etc in closed configurations only. This slide parallels the earlier

“propeller” open trimer slide.

</li></ul>

X ray crystalography<br><br><ul><li>

A simplistic schematic of how an X-ray beam imprints

a 3-D electron “image” of the atomic shape of a protein. The density map on the

right has coordinates for every atom in every amino acid in the folded protein,

but can only be “solved” if the resolution of these atoms is high enough (next

two slides).

</li></ul>

tangier disease<br><br><ul><li>ABCA1, a typical ABC transporter with the full range of genetic lesions

(substitutions, insertions, deletions) that give rise to various levels of

severity of this disease.

</li></ul>

monomer<br><br><ul><li>

A structural map of a single NBD cassette showing the main conserved

motifs. Note that the Walker A &amp;amp; B sequences are very near ATP (yellow-red

stick figure) and Mg2+ (not shown). The A &amp;amp; B motifs help to hold

and coordinate the ATP within the catalytic site. The Q- and H-loops help to

complete the site, with their side chain groups engaged in non-covalent binding

of the coordinated Mg2+ and ATP within the site.

</li></ul>

<br><br><ul><li>

Our MD work at UTS has allowed the

capture of this important h-re-hydrolytic

state in an ABC ATP binding cassette catalytic site. Note the position of side

chain groups that help to cradle ATP in the site, and the ubiquitous Mg2+ that

uses its positive charge to draw negative charge away from the electronegative

terminal phosphate oxygen. Glutamine (in the Q-loop) and histidine (H-loop)

have positive side groups that assist M2+ g in drawing the negative oxygen charge

away, thereby weakening the last phosphate (O-P) high energy bond. This

covalent bond “stretches” as the negative charge weakens and eventually breaks,

releasing the terminal phosphate and ADP. The site opens because the terminal

phosphate is no longer tethered to Mg and the other side groups and the

nucleophilic water. Our MD simulation

captured this water in the correct in-line approach for it’s lone oxygen

electron pair to draw attack the positive phosphate nucleus, which assists in

stretching the P-O bond. The water cannot even approach the phosphate (+ve)

nucleus unless the Mg2+ and side groups draw away much of the negative

charge on the oxygens around the last two phosphates. This

pre-hydrolysis state has never been seen in any crystal structure to date and

therefore emphasises the

power of making the protein move through different energy landscapes. Glutamate

(G481) is the catalytic base that temporarily accepts the proton from water

after ATP hydrolysis. Not shown is a conserved lysine (positive side group) in

the P-loop (Walker A motif) that also helps to draw charge away from the oxygens.

</li></ul>

propeller<br><br><ul><li>.

The open propeller type is unlikely to occur compared

with the closed trimer, as the propeller has too much surface area and

“captures” too much water around the surface according to the free energy

equation</li></ul>

trimer<br><br><ul><li>

The

closed trimer has a smaller outer surface area with more of the surfaces of the

monomers “buried” and in contact with one another (blue arcs).

</li></ul>

cons<br><br><ul><li>

Another major drawback of the open

configuration is the fact that by not being closed, other subunits may be added and this could not possibly

give rise to a stable dimer in this example (two yellow monomers). Such an

open, unstable dimer is exactly as seen in the HisP dimer (previous slide).

</li><li>

Bringing the functional aspects of

structure into play, we se that the Walker A &amp;amp; B motifs in each monomer of the open HisP

dimer are within the ATP site as expected. We also see that the third motif,

the so-called ABC signature motif ‘C’ is remotely located on an outer arm of

each monomer.

</li></ul>

rotational symmetry

<br><br><ul><li>

The UTS team completes and publishes the rival ABC HisP dimer configuration using modelling to

bring all elements of the structure together from

basic folding “rules”, namely: (i) Subunits

formed closed structures with minimum protein surface contact with the water

shell and maximum buried protein surface (with as many amino acids folded

within the core and out of contact with water. This is the most energetically favourable

conformation of closed subunit assemblages. (ii) Sequence conservation “solves”

the problem of the very highly conserved ‘C’ signature motif and its now

obvious interaction with ATP within the catalytic site, along with the other

two (A &amp;amp; B) motifs that are known to bind ATP. The fact that the C motif is

added to the ATP site from the opposite monomer (see above) was a problem that

confused researchers for many years; and which was even more problematic

following the publication of the HisP dimer in Nature in 1998. The closed dimer above also

solves the known allostery of ATP-binding cassette, that is, two ATP sites

working in concert, cooperatively and alternately not unlike a two-stroke

engine, in which the efficiency of the energy output is reliant on alternating

cooperativity. Such cooperativity (allostery) is a common enzymatic mechanism

in hundreds of proteins across all major superfamily classes.

</li></ul>

<br><br><ul><li>

The correct assignment of the alpha-carbon backbone trace

(black lines, central panel) and amino acid side chain groups (right panel)

along the carbon skeleton backbone. This is how a good X-ray imprinted electron

density may is resolved with the accompanying Fourier software.

</li></ul>

resolving side chains<br><br><ul><li>

A covalent bond is about 1 angstrom and if the X-ray resolution is more

than 3 angstroms, the side chain positions have to be built by homology

modelling

</li></ul>