Lecture 10 – Proteins Secondary, tertiary, and other structures

Prions

• Proteinacious infectious particles

• We think of almost all agents that cause infection to alive (bacteria, unicellular eukaryotes, etc.), or at least mimic a living organism (virus)

• Prions are completely different

Are Prions infectious?

Yes

◦ They’re infectious, even though they’re just a protein. No nucleic acid.

◦ They don’t “replicate” so much as cause your own normally folded proteins to become misfolded

Important to distinguish: They’re infectious, but very difficult to transmit ◦ i.e. I probably can’t sneeze infectious particles onto you

How does a generic prion disease work?

Prion proteins exist as normal proteins in normal cell membranes

Through either spontaneous misfolding, genetic abnormality, or introduction of infectious proteins, there is an over accumulation of unfolded and non-digestible protein matter

◦ Can’t be targeted by ubiquitin and subsequently removed by proteosomes ◦ Can’t be degraded effectively in lysosomes

How are prions infectious?

PrP(C) is the normal variant.

◦ When it comes into contact with the infectious version PrP(Sc) , this version changes the normal protein’s shape into the infectious version

◦ Now there are two infectious particles! Continue process over and over…

How many codified prions diseases are there in humans?

In humans, somewhere between 9-10 different diseases, but occur through three main routes

◦ Spontaneously developed

◦ Genetically derived ◦

Due to infectious particles

◦ All are relatively rare

The more interesting prion diseases:

◦ Kuru

◦ sCJD – Spontaeous Cruetzfeldt-Jacob disease

◦ Also has a familial version

◦ FFI - Fatal familial insomnia

◦ Also has a sporadic version

◦ Iatrogenic CJD

Kuru

Probably the most well known… ◦

Ritualistic Cannibalism – of the brain

◦ Suspected to be caused by one spontaneous case of CJD… ◦

Then, their brain was eaten, spread to rest of group

Spontaneous Cruetzfeldt-Jacob disease

The most common codified prion disease

FFI - Fatal familial insomnia

◦ Similar symptoms, but includes bouts of insomnia, which ultimately cause the death of the patient

Iatrogenic CJD

Derived from infectious particles left of medical equipment

General pathology of prion diseases

PrP(C) → PrP(Sc) , then what?

◦ PrP(Sc) accumulates…

Beta sheets fold themselves into beta helices

These “beta helices” then form fibrils, long strands of non-degradable proteins

Tissue pathology

Vacuoles – The typical damage that occurs in the disease

◦ Hence – “spongiform”

The void regions form from cells that have died, having left an empty region in its place

Tissues in prions diseases

• Spongiform degeneration

• Prp plaques (formed by over accumulation of protein structures)

• Gliosis

• Florid Plaque

Behavioural effects of general prion diseases

Cognitive dysfunction is the most well known, and includes

◦ Irritability and anger

◦ Delusions

◦ “as if someone is taking over my mind”

◦ Hallucinations

Eventually, after so much damage to the brain has occurred, patients pass within 1-2 years of diagnosis

How do prion diseases occur?

Many of these diseases can happen spontaneously, they are poorly understood, and completely incurable

Primary structures of proteins:

◦ The string of polypeptides coded from DNA, potentially modified post-translation, and the subsequent sulphur bridges (covalent bonds) that form within the strand

Secondary structures of protein

Local repeating structures that form once the final polypeptide sequence is complete

◦ Typically formed via hydrogen bonding between N and O groups, and occasionally C groups, in the amino residues

Tertiary structures of protein

The final three dimensional structure that forms once the repeating structures are in place

Quaternary structures of proteins

Multiple fully folded subunits coming together to form complexes

Bond Angles in proteins

Φ =

Ψ =

Φ = phi Bond of N group to C alpha

Ψ = psi Bond of C alpha to C-O

Secondary protein structures What are included in these groups?

Our two most common:

1. Alpha helices (right handed)

2. Beta sheets

Next in line:

3. 310 helix

4. Polyproline II (or polypeptide II) helix

5. Left handed alpha helices

Alpha helices, how are they formed?

Hydrogen bonds formed between:

◦ Oxygen of carboxyl group and nitrogen of amino group

◦ Side chains (R groups) stick out perpendicular to the helix

◦ Depending on which groups are present, the helix could be hydrophilic, phobic, or amphipathic.

◦ When we refer offhand to alpha helices, we are almost always referring to right-handed helices

Beta Sheets: Anti-parallel vs. parallel sheets

Anti-parallel: N terminal and C terminal of the cross-bonded sheets run in different directions

Parallel: N terminal and C terminal of the cross-bonded sheets run in the same direction

Bonds between sheets are formed between one of

1. The nitrogen atom of the amino residue with the oxygen of the carboxyl group or

2. The oxygen of the carboxyl group with the beta carbon of the other side (less common)

3₁₀ helix

 the more uncommon helix

◦Typically, the third most common secondary structure found in amino acids

◦Hydrogen bonds are formed between nitrogen group of prolines or amino groups and a carboxyl group

◦Why 3₁₀?

◦3 Amino groups per turn of the helix, 10 atoms found between the hydrogen-bonded atoms

 Polyproline II helix

◦No stabilizing hydrogen bonds between turns

◦Composed of primarily proline amino residues, with some glycine

Left-handed alpha-helix

 the most uncommon helix!

◦Rare, considerably less stable than its right handed version

How do we know which amino acids exist in which structures?

 Ramachandran Plots

How do  Ramachandran Plots work?

◦Because we know the predicted bond angles induced between the N-Cα (phi) and the Cα-COO (psi) bonds by the five specific secondary structures of all of the amino acids, we can isolate these structures in proteins via crystallography

◦They give us plots of which amino acids are present in the secondary structures

steric clashing

 We also know that some bond angles are simply not possible

This is steric clashing that occurs between the residues of the amino acids

What is a sterically non allowed conformation?

Φ =0, Ψ =0 is not allowed in any polypeptide chain because of the steric crowding between main-chain atoms. A tripeptide is shown, where the central amino acid has Φ =0, Ψ =0

The carbonyl oxygen of residue #1 would clash with the steric residue of the amide hydrogen of residue #3

Ramachandran plot

A map of this type can be used to describe the backbone conformation of any polypeptide residue as well as the secondary structures of proteins.

The coordinates are the values of the bond angles ϕ and ψ,

The white areas correspond to sterically allowed conformations

The gray dots in the background show the observed ϕ and ψ angles for over 10,000 alanine residues found in protein X-ray crystal structures.

The circles withsymbols correspond to the secondary structures

B strand ϕ and ψ angles

ϕ -150 to -100

ψ -120 to 160

Alpha helix ϕ and ψ angles

ϕ -70 to -60

ψ -50 to -40

3₁₀ Helix ϕ and ψ angles

ϕ -70 to -60

ψ -30 to -10

Polypetide II helix ϕ and ψ angles

ϕ -80 to -60

ψ 130 to -160

Ramachandran plots for glycine and proline.

Glycine has the greatest number of allowed ϕ, ψ angle combinations, whereas proline has the fewest.

 When we think of proteins, we often think:

◦Enzymes

◦Extracellular to intracellular signalling

◦Secondary messenger signalling

◦Hormones and hormone signalling

◦Compound transport

 These are almost always globular proteins, which include tertiary and sometimes quaternary structures

 Other protein structures can be finalized for their main purpose with, mostly, only secondary changes

Fibrous proteins

amino acid structures which are elongated “strings” of polypeptides

 The three main fibrous proteins:

◦Keratin

◦Fibroin

◦Collagen

What amino acids are found in fibrous proteins?

 Usually, these proteins have AAs with small, and uncharged, R-groups, which can include:

◦Glycine

◦Alanine

◦Serine

◦Proline

 With other amino acids being present in varying numbers depending on the exact nature of the fibre.

Alpha (α) and Beta (β) keratins

•Alpha keratins

•Compose the hair and fingernails of mammals

•Beta (β) keratins

•Talons, beaks, feathers

alpha (α) Keratin

Common structural protein – Approximately 300 residues long

Composed of a polypeptide that is almost entirely an alpha helix, hence the name

Two of these strands come together and intertwine, due to some of their small and hydrophobic side chains (glycine, serine)

The larger side chains point outwards

Beta Keratin

A continuous strand of proteins, but rather than alpha sheet being the main structures, it’s the beta helix

Less important for mammals…

More important for other vertebrates:

Helps form the feathers, beaks, and claws of birds and/or reptiles

Fibroin

 Again, another secondary structure less important for mammals, but more for invertebrates

◦Silk worms and spiders

 Composed almost entirely of anti-parallel B-sheets aligned one on top of each other

◦Again, composed of small amino acids

◦Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-Ala-Gly-Ala-Gly)₈

◦Slightly different patterns can allow for different structural properties

Structural unit of fibroins

• Stacked b sheets. alternating layers of interdigitating adenine or glycine side chains

• Fibres are very flexible because bonding betweens the sheets involves only the weak van der waals interactions between side chains

Collagen

 Makes up the largest components of the fibrous proteins in animals

◦Connective tissue holding skin together, forms the matrix for holding together bone, and is an important connective piece between bones (tendons and ligaments)

It’s also (sort of) what you’re eating when you have Jell-O

 How is collagen composed?

◦A triple helix of intertwining polypeptides

◦A repetitive string of amino acids composed of primarily

◦Glycine, Proline, and a modified version of proline (-4-hydroxyproline)

Gly-X-Y àGly-X-Y à etc. etc

 However, variants in this pattern can result in different collagen structures

vitamin C

The enzymes that catalyze the hydroxylations of proline and lysine residues in collagen require vitamin C, L-ascorbic acid

important for collagen

The structure of collagen fibers.

The basic unit of the collagen fiber is a triple helix of three polypeptide chains

how much fibrous proteins are in an organism?

 the amount of fibrous proteins in an organism is relatively small…

 Globular proteins compose the vast majority of proteins in organisms, including humans

What are Globular proteins involved in?

 These molecules are involved in enzymatic and transportation work, and can include proteins required for/as:

◦Signalling (insulin)

◦Anabolism and catabolism (enzymes in metabolism)

◦Signal receptors (neurotransmitter receptors)

◦Destruction / degradation of unnecessary / harmful substances (typically enzymes or antibodies)

Examples of diverse globular proteins

Myoglobin- Transport

Neuraminidase- Enzyme

Triosephosphate isomerase - enzyme

How do we classify globular proteins?

1.The types of secondary structures present

I.Alpha helices, beta sheets, 3₁₀ helix, etc.

2.The number of domains present

I.A specific  tertiary 3D structure, typically useful for transport or enzymatic activity

3.The types of repeating patters present within the domains

I.i.e. types of a-helices and b-sheets present

What is this?

A three-dimensional model of the stacked B sheets of fibroin showing alternating layers of interdigitating alanine or glycine side chains.

Three sheets are shown with H atoms in gray, N atoms in blue, O atoms in red, and C atoms in either green or magenta.

Fibers are very flexible because bonding between the sheets involves only the weak van der Waals

interactions between the side chains.