Lecture 7: Protein Structure and folding

1. Primary structure: amino acid and genetic code

The 22 amino acids found in proteins

• proteins are chain-like polymers of AA

• Each AA has amino/ carboxyl group

• diff R groups

• pH 7 = charges on backbone

  • PH2= +1 change

  • PH7= 0change

  • Ph12= -1 charge

AA to remember

Lysine, Arginine, Histidine, Pyrrolisine (Basics)

N terminal has lots of Lysine, adn arginine, that are modified and are epigenetic modifications

Histidine is fron histone- interact with DNA

Pyrrolisine (modified lysine)

Aspartic acid (asparagous), glutamic acid (acid)

Tyrosine- phosphorylated adn dephosphorylated

Cysteine (created disulfide bonds) for protein protein interactions (P)

Methionine (s-group) (NP)- first AA in eukaryotic and codon is AUG

selenocysteine (modified cysteins)

AA

• AA help by petide bonds due to condensation reaction

• peptide= sequence of AA

  • has partial double bond character due to resonance

  • free rotation occurs between a-carbon and peptide unit

  • trans and cis config possible due to rigid peptide bond

AA cont

Translating genetic code

• DNA seq read in triplets using antisense (non-coding) strand as a templet that directs synthesis of RNA via complementary base pairing

• ORF (open reading frame) in mRNA indicates presence of start codon followed by codons for a series of AA and ending with termination codon

The genetic code

• codon box

  • made of 4 three letter codes (64 in all)

  • 61 codons recognized by tRNA for incorporation for the 20 common AA

  • 3 codon signal termination or code for selenocysteine and pyrrolysine

The genetic code is degenerate

• tRNA is specific to specific AA recognize multiple codon triplets that differ in 3rd letter only

• Pairing for first 2 codon position are the same for codon and anticodon using complementary base pairing rule

• wobbles” (non-Wat/crick base pairing) occue at third position

look at graph

3rd of codon and 1st on anitocodon has flexibility

Genetic code is not universal

In certain organisms and organelles the meaning of select codons has been changed.

The 21st and 22nd genetically encoded amino acids

• UGA code for selenocysteine found in (UGA used as codon to expresses selenocysteine)

  • 15 genes in prokryotes involved in redox reactions

  • 40 genes in euk code for various antioxidants and type 1 iodothyronine deiodinase

• UAG code for pyrrolysine found in(UAG used as codon to expresses pyrrolysine)

  • some archaebacteria and eubacteria

Codon bias

• freq at which different codon are used vary between diff organisms and between proteins expressed at high or low levels within same organism

• codon bias can have major impact on effeciency of expression of proteins if they contain codons that are rarely used in desired host

D- and L-amino acids in nature (na)

• enantiomers

• living org maininly have L-AA

• ribosomes use L-AA to make proteins

• D-AA made in pathways that do not involve ribosomes

  • can be made by L-AA by post translation process

2. The three-dimensional structure of proteins

Dalton (Da)= describes molecular wieght of proteins

• most polypeptide chain habe molecular weight of 20-70kDa

• avg mol/weight of AA is 110

2nd structure

• stabilized by H-bonds (amino is donor, and carb is acceptor) that ceate a peptide bond

• structures

  • a-helix

    • Prolein (helix breaker) cannot act has a donor in H-bonding

  • B-pleated sheet

    • stab by H0bind

    • Parallel

      • same directions

    • Antiparallel (more stable)

      • opp directions

  • unstructured turns

    • turns connect a and b in proteins

    • short loops that do no exhibit a defined secondary structure

tertiary structure

• mostly stabilized by noncovalent bonds:

  • hydrophobic interactions

  • H-bonds

• Primary covalent bond within/between polypeptide are disulfide bridges between cysteins (S-S)

• structures

  • globular proteins

    • most proteins are spherical

  • fibrous proteins

    • long filamentous or rod-like structures

  • membrane proteins

    • differ from soluble proteins in relative distribution of hydrophobic AA residues

    • 7 transmembrane helix structure is a common motif in membrane proteins

Quarternary structure

• 1 or more polypeptide subunits creates functional protein

• great versatility of function

3. Protein function adn regulation of activity (na)

• proteins made of domain with specific function

  • each domain made of continous AA sequence

Ezymes are biological catalysts

• lower activation energy → speed up reaction

• substrante bind complex with enyme by binding to active site

• induced-fit mechanism

Regulation of protein activity by post-translational modifications

• post translation mod

  • ex: phosphorylation adn allosteric effectors

• After translation- proteins joind cov& noncov to other molecules

  • exL lipoproteins, phycoproteins

• reversible phosphorylation of a AA side chain is most common regulatory mechanism

Protein phosphorylation (NON remember)

• can cause protein to change shape

• phosphorylates side chain can be part of motif to facilitate formation of a multiprotein complex

• phosphorylated side chain can promote dissociation of a multiprotein complex

AA: Ser, Thr, Tyr

• Kinase catalyze addition of phosphate group

  • 2 protein kinase groups

    • phosphorylate serine/threonine side chains

    • phosphorylate tyrosine side chain

• phosphatase remove phosphate

  • less specif

Allosteric regulation of protein activity

• ligand induces conformational change

• an active site/ another binding side is altered in a way that increases or decreases activity

Cyclin-dependent kinase (CDK) activity is regulated by both allosteric modification and phosphorylation

• Partial activation of CDK

  • binding cyclin to CDK causes conformational change

    • T loop moves away from entrance of active side

• Fully activation of CDK

  • phosphorylation of THr160 in T loop by CDK-activating kinase (CAK)

  • stabilizes active site (catalytic cleft)

4. Protein folding and misfolding (No remember)

• protein folding is innitiated before completion of protein synthesis

• other protein go major olding after reease into cytoplasm or organelle

Molecular chaperones

• Increase efficiency of protein folding.

• Reduce the probability of competing reactions such as
  aggregation.

• Heat-shock proteins promote protein folding and aid in the destruction of misfolded protein.

  • e.g. Hsp40, Hsp70, Hsp90

Endoplasmic Reticulum (ER) quality control

• secreated protein are translocated into ER

• folding happens before secretions go into Golgi apparaturs

• incorrectly folded proteins are directed by unfolded protein response and targeted for degradation

Ubiquitin-mediated protein degradation

• Ubiquity (a 76 amino acid polypeptide) is attached to a protein by a series of enzyme-mediated reactions.

• The ubiquitin-conjugated protein is then targeted to the 26S proteasome.

• Ubiquitin is released and the target protein is degraded
  by proteases

ubquity activated by atp (E1) activating enzyme

Ubiquitin-conjugating enzyme(E2) congregate enzyme

Ubiquitin Protien ligase (E3) ligase enzyme

Protein misfolding diseases

• Formation of protein aggregates is linked to at least 20 different human diseases

• Amyloid or amyloid like fibrils: normal soluble proteins accumulate as insoluble deposits

  • these proteins in amyloid-like fibrils fold into cross B-spine

Prions

• Primary cause of transmissible spongiform encephalopathies (TSEs)

• progressive neurodegenation

• dementia

• loss of muscle control of voluntary movements\

  • No cure, death 6monts 1 year

• Animal forms

  • scrapie (sheep)

  • BSE(mad cow disease) bovine spongiform encephalopathy

  • chronic wasting disease (elk/deer)

The “prion only” hypothesis of infection

• Stanley Prusiner: Nobel Prize in 1997.

• Lack of immune response characteristic of infectious diseases.

• Long incubation time (up to 40 years for kuru).

• Resistance of the infectious agent to radiation that destroys living microorganisms

  • (e.g. viruses, bacteria)

  • radiation destroryes DNA/RNA

• infectious agent is a protein called PrP^Sc that can replicate self within the body

• normal cells have normal PrP^c , the PrP^Sc has same AA sequence but prion is misfolded into diff 3D structures

• in infected cell, host protein PrP^c misfolds to form new prions called PrP6Sc

• can survive sterilization techniques