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BE 167L Lecture 3 General (ppt)

BE 167L Lecture 3 General (ppt) - Lecture 3 • ...

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Unformatted text preview: Lecture 3 •  Fluorescence and fluorophores •  Reac4vity of Amino Acids •  Standard Bionconjuga4on Chemistries •  Example: ACaching Fluorophores to Proteins •  Next Lecture – Polymeriza4on – SoG lithography – Micro ­contact Prin4ng Fluorescein DAPI - 4',6-diamidino-2-phenylindole Rhodamine Key point: fluorophores in visible wavelengths are highly conjugated Photons are absorbed Electrons are vibrationally excited They relax to lowest excited state They emit a photon of lower energy Franck-Condon Principle – No molecular movement during photon absorption Relaxation is faster than emission, leading to the Stokes Shift Quantum yield, ratio of number of photons emitted to absorbed, Rate of radiative decay, Γ Non-radiative decay, knr •  Solvent dielectric proper4es •  Temperature •  Nearby molecules affec4ng energy of excited or ground state •  Neighboring absorbing molecules (Resonance Energy Transfer – RET or FRET) •  Quenching – Oxygen and other electronega4ve molecules, aCenua4on by absorp4on. The opposite of this is also possible by bringing a quenching molecule close to the fluorophore Fluoresc. Recovery after photobleaching (FRAP) Key Concept: Photobleaching results from reaction of fluorophores, usually with triplet-state oxygen Key Concept: Avoid photobleaching by limiting exposure and/or intensity of light, adding “anti-fade” agents that limit oxygen reactivity. Amino Acids and proteins: a quick primer Key to acceptance: excitation/emission overlaps with fluorescein: can use same filter set Fluorescent proteins: uses in bioengineering as reporter genes •  Visualize outcomes of experiments •  Real ­4me data output from in vivo experiments •  Control func4ons of certain cells or systems for experimenta4on •  Make fluorescent pets Discosoma sp. Directed Evolution of Red Fluorescent Protein (Roger Tsien, UCSD) • matures more completely, • more tolerant of N-terminal fusions • over tenfold more photostable. FP are available in many colors with different proper4es •  FAD Several fluorescent molecules common to biological specimens –  –  –  –  –  –  –  flavins, flavin proteins and nucleo4des (FAD and FMN), B vitamins, reduced pyridine nucleo4des (NADH and NADPH), faCy acids, cytochromes (cytochrome P450, heme, etc.) porphyrins, serotonin, and catecholamines. Vitamin A NADH heme –  In plants serotonin –  chlorophyll (red fluorescence) and lignin (green fluorescence). In general, autofluorescence emission is greatest when live cells are examined with blue and ultraviolet excita4on wavelengths. chlorophyll •  Proteins/Amino Acids •  Nucleic Acids (DNA/RNA) •  Sugars •  FaCy Acids Peptide (Amide) Bond At phyisiological pH, amine is protonated, carboxylic acid is deprotonated Relatively non-polar and hydrophobic side chains, Typically hidden inside protein structures, so methionine isn’t that useful. Again, non-polar. Tryptophan is reactive, but is typically inaccessible. Hydrophilic and accessible, but not very reactive. Sugars are naturally found attached at arrows in glycoproteins, enzymatically derivatized. Key Concept: Most useful for bioconjugate chemistry •  Using nucleophilicity of side chains Key Concept: Order of nucleophilicity determines relative reactivity Microenvironment strongly influences pKa •  Cysteine and lysine undergo high yield nucleophilic subs4tu4on reac4ons at only slightly basic condi4ons •  You cannot select reac4vity using only pH Key Concepts: • Proceeds through active acylated intermediate • End product stability to hydrolysis Amide > Ester > Thioester Key Concepts: •  irect nucleophilic attack D • Formation of amide bonds proceeds through tetrahedral intermediate Key Concepts: • Acts as a nucleophile • Disulfide linkages (redox reactions) •  Determine the availability of amino acids on the protein – Are these amino acids on ac4ve sites of the protein? – Are these amino acids exposed for reac4on or buried? •  Protein structure •  Protein data bank – www.pdb.org Lysine residues on fibronectin N ...
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