Proteins: Form, Function, and Pathology Adrian Poniatowski StudyAid Biochemistry Seminar March 2, 2019 Biochemical Origami The Structure of Proteins

• From primary to quaternary structure • Pathologies are caused by misfolded proteins which are destabilized by seemingly minor amino acid changes • Form strictly defines function in the protein world • Tertiary structure will often be most clinically significant Primary Structure

• Sequence of AA in protein • Creates robust bond resistant to • Read from N- (amino) to C- enviro chgs & flexible for folding (carboxyl) terminal • Dehydration synthesis • Peptide bond: covalent bond • Determines all higher structures between -NH3 (amino) and – COOH (carboxyl) groups α Helix (1 PP chain) Secondary Structure β Sheet (2+ PP chains)

• α helix most common (3.6 AA/turn) • Stabilized by H-bonds b/w carbonyl O’s and amide N’s • β sheets can be parallel or antiparallel to each other • β sheets always have R-hand twist & form core of globular proteins Tertiary Structure • Hydrophobic side chains in interior, hydrophilic outside • Disulfide bridges between sulfide cont side chains provide covalent bond that provide max stability • H-bonds, ionic bonds, hydrophobic interactions contribute to architecture • Domains = fxnal 3D protein units • Denaturation affects 3-ary & 2-ary structures Protein Modification Folding & Denaturation • Chaperone proteins (HSP’s) sometimes coordinate the folding process • Denaturation is irreversible in proteins folded by HSP’s Ubiquitination • Regulates intracellular degradation of protein • Addition of one or more ubiquitin molecules to protein localizes it to proteasome for digestion Review Question

Damaged cytosolic proteins are labeled with (1) and then are degraded by a protein complex called (2): A. (1) ubiquitin (2) translocon B. (1) oligosaccharide (2) chaperone C. (1) acetyl group at specific Lys residues (2) lysosome D. (1) ubiquitin (2) proteasome E. (1) phospahte at specific Ser or Thr residues (2) chaperone Conformational Pathologies

• Abnormal structure or degradation is responsible for many systemic diseases • Misfolding secondary to non-silent mutation in RNA transcript (Sickle Cell, Glu /chg/ -> Val /np/ sub @ position 6) • Not enough protein made (OI) • Degradation of mutated-fxnal or normal protein (CF, AD) • Non-enzymatic/prion degradation (CJD) Alzheimer’s Disease • Hallmark: senile plaques formed • Hyperphosphorylated from β-Amyloid protein tau proteins form • APP cut by γ- and β-secretase to neurofibrillary tangles form β-Amyloid, which clumps • Microtubule system into plaques collapse • Widespread intracell disruption Systemic Amyloidoses • The abnormal cleavage of other proteins can lead to β sheet deposits in multiple organs • AL amyloidosis caused by deposition of Ig light chains in brain, heart, kidney, etc. • AA amyloidisis caused by Amyloid A deposition in mult organs, assoc w/ Rheum Arthritis, IBD, prolonged infectiom • AD is brain-specific amyloidosis Prions

• Contagious pathogens comprised of small, misfolded proteins that induce abnormal folding of normal proteins • Aggregates of misfolded proteins are difficult to degrade and result in apoptosis and neuronal loss • Resistant to heat stress Prion Diseases

Creutzfeldt-Jakob Disease • Spongiform encephalopathy • Rapidly progressive dementia Can be also inherited and • Ataxia & ultimately death acquired. Kuru secondary to ingestion of prion- • Irreversible & incurable infected tissue. • Caused by accumulation of PrP Review Question A pharmaceutical researcher develops a therapy to treat a protein misfolding disorder. Specifically, a mutation in this disorder leads to abnormal protein folding and subsequent intracellular degradation of the protein before it can reach the cell membrane. With the new drug therapy, a drug corrects the processing and trafficking of the protein, enabling it to reach the cell surface membrane. This therapy is most likely to be helpful in which of the following conditions? • A. Alzheimer disease • B. Creutzfeldt-Jakob disease • C. Cystic fibrosis • D. Phenylketonuria • E. Sickle cell anemia • Cystic fibrosis is caused by ΔF508 mutations in the cystic fibrosis transmembrane regulator (CFTR) protein • This deletion causes abnormal protein folding and failure of glycosylation. • The CFTR protein is then targeted for degradation by the proteasome before reaching the cell surface, causing an almost complete absence from the apical membrane of exocrine ductal epithelial cells. Connective Tissue Proteins Elastin Collagen

• Triple helix • Has 3.3 residues per turn • Composed primarily of small non- polar AA’s • Every 3rd AA is a Gly • Rich in Pro and Lys • Motif: Gly-Pro-X or Gly-Hyp-X • Desmosine interchain crosslinks Collagen Synthesis • Synthesis: translation, usu Gly-X-Y (X,Y are typically Pro or Lys) • Hydroxylation: requires Vit C -> scurvy • Glycosylation: formation of triple helix. Problem here -> osteogenesis imperf • Exocytosis and extracell processing • Proteolysis: procollagen to tropocollagen (insoluble) • Cross-linking: CL’s lysine-hydroxylysine (by Cu-cont lysyl oxidase). Problem w/ cross-linking -> Ehlers-Danlos Diseases of Collagen Synthesis

Osteogensis Imperfecta Ehlers-Danlos Syndrome Scurvy • • Spontaneous fractures • Joint Hypermobility Bleeding gums, bruises • Vitamin C is a necessary • Bone & tooth malform • Hyperextensible, fragile cofactor for the hydroxylation of proline and • Blue sclerae skin lysine residues in collagen. • Mutation in Type I • Mutation in Type V • Vitamin C deficiency results in decreased strength of collagen, insufficient collagen (Type III for collagen fibers and causes synthesis vascular disease) scurvy. Review Question

The length of PCR products may be determined based on: A. The binding of a fluorescent dye B. UV light absorption C. Hybridization with specific probes D. Their migration in gel electrophoresis as compared to standards E. Melting temperature • Comparison of normal and mutant forms of gene PCR Method & Application • Prenatal Dx of inborn errors (e.g. cystic fibrosis) • Bacterial & viral microbio Dx • Forensic analysis and low- abundance DNA sample analysis Review Question Molecular biology techniques are used for the identification of individuals (genetic profiling in forensic medicine, clarifying paternity, etc). Nowadays, the most commonly used method is based on: A. Sequencing of specific genes B. Analysis of the number of short tandem repeats (STR) using PCR C. Electrophoretic separation of the total DNA isolated from cells D. Analysis of the number of repeats within telomeres E. Whole genome sequencing STRs and Forensic Medicine

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Source: Khan Academy Review Question

The two most widely used methods of replication of any specific DNA sequence chosen by the experimentalist are molecular cloning and PCR. The fundamental difference between them is: A. Molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells B. All answers are correct C. Molecular cloning generally uses DNA sequences from two different organisms, while PCR amplifies by copying an existing sequence D. Molecular cloning involves formation of recombinant DNA while PCR amplifies by copying an existing sequence E. Two answers are correct