15

Dana Alsulaibi

Jaleel G.Sweis

Mamoon Ahram

Revision of last lectures:

✓ Proteins have four levels of structures. Primary,secondary, tertiary and

quaternary.

✓ Primary structure is the order of amino acids in a polypepetide peptide. It is

very important as it determines the characteristics of the next levels.

✓ Secondary structure (alpha helix + beta strand) presents the localized

organized structure (helix,sheet,strand,turn,loop…). Secondary structure is

stabilized by the hydrogen bonds between the backbone groups ;where the

carbonyl group is the H bond acceptor and the amino group is the H bond

donor ( R groups have no influence on the secondary structure although

some amino acids may disrupt the structure).

Lecture 15:

Parallel vs anti parallel beta sheets

Anti-parallel beta sheets are more stable than parallel beta sheets.

In anti parallel sheets, hydrogen bonds are formed between the amino acid in the

first sheet and the amino acid directly opposite(above) to it in the second sheet .

This provides stability unlike parallel sheets where each amino acid forms a

hydrogen bond to the left or right amino acid of the opposite sheet(not with the

one directly above it).

(my understanding: carboxyl and amino groups are directly opposite to each

other in the anti parallel order, making hydrogen bonds stronger)

Plus , vertical angles are stronger than bent angles making them more stable .

*β sheets can form between

many strands, typically 4 or 5

but as many as 10 or more

*Such β sheets can be purely antiparallel, purely parallel, or mixed

*Valine, threonine and Isoleucine tend to be present in β-sheets.However, Proline

tends to disrupt β strands

What is a turn(Beta turn or hairpin bend) ?

Turns are compact, U-shaped, secondary and small structures which allow

proteins to take a 3 dimensional structure by enabling them to bend and go back.

These turns are composed of 4 amino acids.

2 of them are Glycine and Proline.

Glycine is small and its r group doesn’t create repulsion so It needs less space.

As for Proline, its rigidity breaks the continuity of the peptide at the turn,

allowing the peptide to make a turn. Turns are also stabilized by hydrogen bonds

which are formed between two amino acids between their the backbone groups(

carbonyl and amino groups).

Super secondary structures

They are multiple secondary structures that are formed together.

motif

domain

Two types

1) Motif( a module) : multiple secondary structures that exist consecutively

in a poly peptide sequence. (no seperation between an alpha helix and

the next alpha helix or beta sheet. They come directly after each other

with other structures in between)

✓ Uses of a motif : helps to determine the protein structure only but

not biological function.

✓ Examples of motifs : Helix- loop -helix

Helix-Turn- helix

*Turns are smaller than loops

Binding proteins

The helix-loop-helix and helix-turn-helix are simple motifs that usually serve as

binding proteins in DNA.

Immunoglobulin module is an example of more complex motifs.

Immunoglobulins are proteins that are produced by immune cells .They have very

complex structure that is mainly composed of beta strands and sheets . Having a

Y shape, the upper part binds to the antigen.

Tertiary structure

The 3d dimensional arrangement of all amino acids. The area they take and

spatial arrangement they have (in space) after folding into a polypeptide.

Different ways to look at proteins

a) Trace structure: represents the backbone only with no apparent R groups

b) Ball and stick model: the balls represent the location of atoms in space and

their angles

c) Ribbon structure: alpha helixes are represented as ribbons and beta

strands are represented

as arrows which helps determine the orientation of beta

strands(parallel vs antiparallel)

d) Cylinder structure: alpha helixes are represented as cylinders and beta

strands are represented as arrows

e) Space filling structure : occupied by atoms only with no spaces. Helps to

look at the surface of the protein and any present indentation(طعجات و زوايا(

f) Protein surface map : to look at the topography (the outside structure) of

the protein. Especially helps to determine the structure of drugs that can

bind to the protein.

In the exam we

will be asked about

these structures so

we should know

them well.

Doctor Ma’moon possible exam questions

• bring the structure and ask for its name

• Ask the number of alpha helixes and turns in a specific structure

• What is the name of the motif.

Bonds that stabilize tertiary structure

( 4 non-covalent interactions between the R groups which determine

protein structure):

a) H-bonds: occur not only within and between polypeptide chains but

with the surrounding aqueous medium.

b) Electrostatic bonds(charge-charge interaction) : occur between

oppositely charged R-groups of amino acids. Also, it is called a salt

bridge when it present in a protein structure.

Ex, interaction between Na+ ion and Cl- ion is an electrostatic

interaction

Ex, lysine and glutamate by themselves(not residues) they can form

electrostatic interaction. BUT when both are present as residues within

a protein and they an electrostatic interaction then it is called a salt

bridge

* The same charged group can form either hydrogen bonding or

electrostatic interactions.

c) Hydrophobic : the most important one . Hydrophobic interactions fold

toward the core of the protein to hide from water and expose the

hydrophilic parts outside towards the aqueous environment . This starts

determining the protein structure. It is also the most energetically

favorable structure(requires minimum energy)

However, some polar amino acids can exist in the core of functional

proteins= enzymes . These enzymes need charged amino acids in the

core for the reaction to take place inside of them.

Can polar amino acids be found in the interior ?

Polar amino acids can be found in the interior of proteins

In this case, they form H bonds to other amino acids or to the

polypeptide backbone. They important roles in function of the protein

d) Van der waals interactions: there are both attractive and repulsive van

der waals forces that control protein folding. Although they are weak

forces, they are significant because there are so many of them in large

protein molecules.

*the 4 previous interactions determine protein structure.

Stabilizing factors

These factors do not affect the shape of the protein, but only stabilize it,

preventing it from changing shape.

1st factor : Disulfide bonds between two cysteine amino acids. If the

bond was reduced and broken, the structure of the protein doesn’t

change , it only gets destabilized.

The side chain of cysteine contains reactive sulfhydryl group(-SH) which

can oxidize to form a disulfide bond(-S-S-) to a second cysteine.

The cross linking of two cysteines to form a new amino acid called

cystine

2nd factor : metal ions. These are non protein groups that can form

either non covalent or covalent interactions with amino acids

Non covalent examples: zinc with histidine, myoglobin/hemoglobin with

iron(heme).

Covalent bond examples : iron with histidine of the

myoglobin/hemoglobin

• Function of previous non protein groups(stabilizing factors) :

a)Stabilize structure of proteins

b) Provides the protein with its function

Super secondary structures

They are meltable secondary structures that are formed together.

motif

domain

Two types

2) Domain:

Characterists of domains-

• Domains are larger than motifs, they are composed of hundred of amino

acids residues in various combinations of(alpha helices, neta sheets, turns,

and randon coils).They are also associated with function and structure of

protein.

• If two proteins share a domain,these proteins have a similar function.

• Domains can fold independantly from the rest of the protein.If the domain

was cut from its original protein,it will maintain its shape.Genetic

engineering consists of grouping 2 or more domains to make a final desired

protein

• domains determine structure+ function of proteins

• domains may also be defined in functional terms:

1-enzymatic activity

2- binding ability (e.g a DNA-binding domain)

Properties of proteins :denaturation and renatururation

1) Denaturation

= unfolding of the protein by breaking of non covalent

interactions(disruption of the native conformation of a protein)

Complete denaturaion of a protein can only be achieved by applying a

reducing agent to reduce and break the disulfide bonds. Otherwise, the

protein can regain it’s shape because parts of it are still connected to

each other.

Generally, the denaturated protein will lose its properties such as

activity and become insoluble.

Denaturing agents

➢ Heat : heat breaks up Van Der Waals interactions by increasing

kinetic energy and destabilizing atoms

➢ Extremes of pH: breaks H-bonds and electrostatic interactions.

different Ph values effect protonation and deprotonation according

to pka and thus effects the charges of amino acid side chains

➢ Detergents: disturb hydrophobic interactions. If a protein was put in

a hydrophobic region it will flip to expose its hydrophobic regions

and that may lead to its denaturation.

Detergents

e.g: Triton X-100(non-ionic, uncharged)

sodium dodecyl sulfate(SDS, anionic, charged)

ionic

Non ionic

Denature the protein + add a charge

➢ Urea and guanidine hydrochloride disrupt hydrogen bonding and

hydrophobic interactions.

➢ Reducing agents such as β-mercaptoethanol (βME) and

dithiothreitol (DTT). Both reduce disulfide bonds. These do not

denature the protein but destabilize it.

2)Renaturation

➢ Renaturation is the process in which the native conformation of a protein is

re-acquired .

➢ The protein can fold back to its original structure by removing the

denaturing factor and reducing agent. However mostly small proteins can

do that. Large proteins need help.

➢ Renaturation can occur quickly and spontaneously and disulfide bonds are

formed correctly.

Factors that determine protein structure –

The least amount of energy needed to stabilize the protein. This is determined by:

– The amino acid sequence (the primary structure), mainly the internal residues.

– The proper angles between the amino acids

– The different sets of weak noncovalent bonds that form between the mainly the

R groups.

– Non-protein molecules.

Can an unfolded protein re-fold?

If a protein is unfolded, it can refold to its correct structure placing the S-S bonds

in the right orientation (adjacent to each other prior to formation), then the

correct S-S bonds are reformed. This is particularly true for small proteins.

The problem of misfolding

When proteins do not fold correctly, their internal hydrophobic regions become

exposed .trying to hide from water they interact with other hydrophobic regions

on other molecules, and form aggregates and clusters.

Chaperons

As we said before, large proteins need help in folding, this is done by chaperons(

الرفيق/المساعد ) and will be discussed in the next sheet.

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