Proteins

Ocular Biochemistry

What are polymers of amino acids linked together by?

peptide bonds

Molecular weight < 10k

peptides

Molecular weight > 10k

proteins

How are proteins related to genetic expression

they are the end products

What are proteins composed of?

at least **one carboxylic acid**, and **one amino group** attached to an adjacent ==alpha-carbon==

What are bigger, peptides or proteins?

Proteins

Why are proteins good buffers?

Have a great balance of charge due to their structure

What is transcription?

Making an mRNA copy of a DNA strand

What is translation?

Using an mRNA to make a protein

General functions of proteins

* mechanical support
* regulate growth and differentiation
* transportation & storage
* catalysts
* motion
* nerve propagation and immune protection
* intra/extra cellular buffers

What structure is A? B?

A: peptide

B: protein (more complex, larger)

Functions of proteins in the eye

* support structure and clarity of cornea
* variable light refraction of the lens
* initiative transduction of light into electrical signal
* generate IOP
* lyse bacteria in precorneal tear film

Solubility classifications of proteins

* water-soluble
* lipid-soluble
* insoluble

Unsubstituted amino acid

glycine

Dicarboxylic amino acid

aspartic acid

Diamino amino acid

Lysine

Amido amino acid

Asparagine

Hydroxy amino acid

serine

Aromatic amino acid

phenylalanine

Sulfur amino acids

cysteine

Cyclic amino acid

Proline

What happens when proteins are denatured?

can lose their function temporarily or permanently

What happens when proteins are denatured?

change in conformation of the native form of the protein

What are causes of protein denaturation?

* change of temperature
* ionic composition
* environmental factors

What type or proteins are more prone to denaturation?

Catalytic proteins

What are the 4 basic structures of proteins?

1. protein
2. secondary
3. tertiary
4. quaternary

What is a primary protein structure?

chain of amino acids

What is a secondary protein structure?

When sequence of amino acids are linked by **hydrogen bonds**, creating pleated sheets or alpha helix

What is the structure of a tertiary protein?

when there are attractions present between pleated sheets/alpha helix (secondary protein)

What is the structure of a quarternary protein?

consisting of more than one amino acid chain

How many configurations/shapes does each amino acid sequence assume?

4

Why would a protein change in configuration?

* bulkiness
* change in density
* hydrophobic regions of amino acids

What determines the shape of a proteins?

* hydrogen bonding
* disulfide bonding within and between amino acid chains

antiparallel

sequence parallel to another sequence, but with opposite alignment

What are the types of structures of secondary protein?

1. random coils: irregular forms
2. Beta-turns: 180 turns that connect beta-pleated sheets
3. Beta-pleated sheets: parallel or antiparallel sequence of primary structures

What are the structures of secondary proteins held together by?

numerous hydrogen bonds

What is a domain?

specialized region in a protein having specific function

What is a motif?

simplified version of a domain

What structure is most common in soluble and transmembrane proteins?

alpha-helix structure

what are alpha-helices and beta-pleated sheets stabilized by?

hydrogen bonds

What type of structure is Rhodopsin mostly?

alpha-helical

What do quarternary structures of proteins consists of?

two or more polypeptide chains

Describe an immunoglobulin structure

* 3D
* 2 light and 2 heavy chains
* **disulfide bonds/sulfur bridges**

Function of lysozyme

lyses the peptidoglycan cell wall of **gram + bacteria in tear film**

Rhodopsin

protein found in the outer segments that mediates visual transduction

Post-translational modification

alteration of the peptide chain of proteins after synthesis

Significance of protein function to the eye

* source for generation of osmotic pressure across cell boundaries
* corneal deturgescence
* generation of IOP

What is visual transduction?

conversion of light energy to electrical energy

What are crystallins

group of structural proteins in the crystalline lens

Where are crystallins found in the lens?

epithelial and fiber cells of the ocular lens

What solubility type of protein are crystallins?

water soluble

Why are crystallins important for our vision?

when they maintain elongated shape, **they affect the refraction of light**

How many types of crystallins are found in the lens?

1. alpha
2. beta
3. gamma

subtypes of alpha crystallins

1. alpha-A
2. A-beta

subtypes of beta crystallins

1. beta-H
2. beta-L

subtypes of gamma crystallins

gamma A-F

Role of alpha crystallins

* molecular chaperones
* maintain the normal molecular conformation
* preventing crystallin aggregation (senile cataracts)
* inhibits light scattering

What are senile cataracts?

newly forming cataracts, when the crystallins are beginning to lump together

What type of crystallin is the best chaperone?

alpha crystallin

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* helps fold and stabilize beta and gamma crystallins

How is crystallin concentration different in cells?

concentration is **twice** that of most intracellular proteins (33% vs 15%)

When alpha and beta crystallins are made, are they stable?

yes, because they are **acetylated**

Why is acetylation important?

prevents cellular depredation of proteins

How can alpha and beta crystallins be altered?

* phosphorylation
* incorporation of sugars
* deamidation/degredation of the polypeptide chain

What is the secondary structure of all human crystallins?

beta-pleated sheets

What structure do some alpha crystallins have?

helical structure

describe the structure of a gamma crystallin

* two sets of motifs that are aligned in a V shaped
* **two domains for each gamma crystallin**
* hydrophilic on outside, hydrophobic inside

Why are **methionine (102)** and **cysteine (109)** important to point out in gamma crystallins?

they are potential locations of oxidation of protein, which can cause opacities in lens

Why does oxidation bad in the lens, and what does it cause?

promotes crystallin aggregation, can cause cataracts

Describe the structure of alpha crystallins

* two bound rings of crystallin subunits laid on top of each other
* large units - hydrophilic
* small units - hydrophobic

Where do the chaperon activities of alpha crystallins occur?

at the extensions that cover the central cavity

How many alpha crystallin subunits are there

40 different subunits

What crystallin(s) is/are found in the lens epithelium

ONLY alpha crystallins

What crystallin(s) is/are found in lens fiber cells?

all three crystallins

Can lens proteins be renewed?

No, they are not metabolically renewed in the lens fiber cells

What are the steps in the transformation process of crystallins when aging occurs

1. phosphorylation
2. disulfide bond formation
3. deamidation
4. peptide bone disruption

What happens during phosphorylation in lens aging?

amount of phosphorylation chains of alpha crystallins increases

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this causes negative charge of the protein to increase

Describe disulfide bond formation what type of process is disulfide bond formation?

oxidative process

Why are cysteine groups important when talking about disulfide bond formation?

cysteine groups are a potential source causing the bonds to form

Why is gamma crystallins the most unstable?

they have the highest concentration of cysteine

What is deamidation?

loss of amide group

What is an example of deamidation in the lens?

aspargine and glutamine are converted to their corresponding dicarboxylic acids

What is peptide bond disruption?

when peptide bonds break in crystallins

Where does the peptide bond break in beta crystallins

near the N terminal region

Where does the peptide bond break in gamma crystallins?

near the C terminal region

What causes peptide bond disruption?

endopeptidase activity

What change in concentrations causes cataracts?

large increase of water soluble protein fraction after age 50

What is the insoluble fraction in an individual 80+ years old

50% or more

In normal aging process, describe cataract formation

* insoluble fraction increases
* disulfide bonding (cysteine), crosslinking

what are high molecular weight aggregates?

aggregated of crystallins

How do we term the HM aggregates of crystallins?

HM1, HM2, HM3, HM4

Which HMWA of crystallins are not considered threats for cataracts?

HM1 and HM2 because they are **soluble complexes**

Which HMWA of crystallins are considered a threat for cataracts?

HM3 and HM4 because they are **insoluble complexes**

Describe HM3

* **held together by disulfide bonds**
* mol wt: 2-33 mil. daltons
* associated with cortical cataracts
* noncrystallin protein

Describe HM4

* **do not involve disulfide bonds**
* associated with dark color of nuclear cataracts

Difference of nuclear cataract and cellular debris formation

formation of disulfide, covalent bonds leads to cellular debris formation

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\*\*\****nuclear cataract related to HM4 which DOES NOT have disulfide bonds***

Cortical cataract mechanism

1. normally folded crystallins protect oxidizable amino acids
2. hydrogen peroxide in aqueous can oxidize methionine and denature protein
3. exposed cysteines oxidize and form disulfide bonds with other crystallins and membrane proteins

Why is glutathione important? Is having less glutathione good or bad?

* protects crystallins from crosslinking by binding exposed groups like cysteine
* less = bad

What is the role of tryptophan?

* forms component of crystallins
* thought to be associated with nuclear cataracts
* **may contribute to the increase in yellow coloration of the lens with age**

What is the influence of UV light on the lens?

* oxidizes tryptophan to N-formylkynurenine
* contributes yellow/brown coloration of nuclear cataracts

What type of cataracts are associated with “nondisulfide crosslinking characteristic of HM4 aggregates”?

nuclear cataracts