Chemical Bio Important info

Acidic AA

Aspartic acid, glutamic acid

Basic AA

Arginine, Lysine

Aromatic AA

Phenylalanine, Tryptophan

Aliphatic

Isoleucine, Leucine, Valine

Polar AA (hydrogen bonds)

Asparagine, Aspartic acid, Glutamine, Lysine, Serine, Threonine

Non polar AA

Alanine, Isoleucine, Leucine, Phenylalanine, Valine

Are peptide/amide bonds rotational?

No

Octahedral splitting

eg above (z² and x² - y²), t2g below

Tetrahedral splitting

t2 above, e below

How long is tRNA?

75-90 nucleotides

Major groove of DNA

Side opposite the deoxyribose

Water soluble proteins

Have non polar cores (hydrophilic on outside of globular proteins)

Membrane proteins

Polar/hydrophilic inside. Non polar outside directly facing lipid bilayer

Zinc finger protein

2 cys and 2 His side chains - S and N atoms that bind zinc due to d10

Iminol or keto form?

Keto

Enamine or imino?

Enamine

Why phosphate?

Abundant, soluble, kinetically stable, trivalent, anionic, durability

Group transfer molecules

ATP, NAD(P)H, SAM, acetyl-CoA

Mustards

DNA alkylators, nucleophilic S/N atoms 2 carbons away from leaving group, cancer treatment, chemical warfare agent (H/HD, Q, HNI)

Stop codons

UAA, UAG, UGA

Molnupiravir

Covid prodrug treatment, incorporates into RNA and can be translated as C or U

Zidovudine (AZT)

Treats HIV/AIDS, phosphorylation and trapping in cell causing chain termination

Huisgen reaction

Uses azides and alkynes to react with heat, DNA tagging

Click chemistry

Copper catalyses reaction, room temp in water, tag with radioactivity or alkyne handles

PCR denaturing

94-94 degrees, heat to break hydrogen bonds and separate strands

PCR annealing

55-65 degrees, cool to bind primers

PCR extension

72 degrees raise temp so DNA Taq polymerase can extend primers and synthesize new strands

DNA ligase

NH2 attacks phosphate to remove diphosphate then new base attacks to remove ligase

Mg-ATP

Divalent Mg2+ shields 2 of side chain negative charges

Phosphoryl transfer

Glucose attacks 3 phosphate to cleave ATP into ADP + Pi, Catalysed by kinases

Nucleotidyl transfer

Attacks 1 phosphate to form pyrophosphate (PPi) favourable reaction, catalysed by RNA polymerase

Pyrophosphoryl transfer

Attacks 2 phosphate to add 2 phosphates. Removes right side of ATP. Uncommon.

Adensoyl/Nucleosidyl transfer

Nucleophilic attack at ribose 5 carbon. Provides SAM synthase. For vitamin B synthesis

S-Adenosyl methionine (SAM)

Positive charged sulfur, used in Wybutosine synthesis (6 SAM). Can add methyl group to membrane as part of maturation

Nicotinamide adenine (phospho)dinucleotide (NAD(P)H)

Redox active molecules NADH from glycolysis and TCA cycle, reduces aldehydes, ketones and thiolesters. powerful 2 electron reductant

NAD+

Non redox, good leaving group

Fatty acids

Long even numbered carbon chains

Acetyl coA

A biotin dependent enzyme

Fatty acid synthases

Ketoreductase, Enoyl reductase, Thiolesterase

Ketoreductase (KR)

Uses NADPH to reduce ketone

Enoyl reductase (ER)

Use NADPH to reduce ketone (conjugate addition of hydride)

Thiolesterase (TE)

Hydrolyzes chain (SACP on end becomes OH)

Cerulenin

Inhibitor of fatty acid synthesis, reacts with active site of Cys in keto acyl synthase to block activity

Allolactose

Binds to repressor and allows RNA polymerase to transcribe

IPTG

Allolactose analogue that maintains expression from lac operon

Automated DNA synthesis steps

Detritylation, addition phosphitylation, capping, oxidation then deprotection and cleavage

Porphyrin

Metalloprotein, binds Fe to make Heme

Hemoglobin (Hb)

Transports oxygen in blood, Dimer of heterodimers, Low/high spin alters structure, cooperativity when one subunit influences another’s binding

Deoxy Hb

Tense state, Fe below plane of ring, High spin

Oxy Hb

Relaxed state, Fe within ring, Low spin

Biotin

Binds to streptavidin

Firefly bioluminescence

Luciferase + ATP → complex → luciferin dioxetanone → s1 state → relaxed state + light

Firefly bioluminescence

Adenylated at carboxyl to form AMP ester

Imaging with chemiluminescence

No laser or background, no filter, no phototoxicity, simplifies microscopy, efficient, high quantum yield

Luminol

Oxidation catalysed by transition metals (iron in blood). Spray with base/H2O2. Blue colour visible as goes to ground state 3-aminophthalate

Chemiluminescent Immunoassay (CLIA)

Enzyme is horseradish peroxidase, substrate is luminol with base + H2O2

Glow sticks

Phenyl oxalate ester, break hydrogen peroxide highly strained ring system breaks, indirect as energy populates the S1 state

Green Fluorescent Protein (GFP)

238 aa chain, naturally occurring, fold up with chromophore inside of 11 strand beta barrel

Jablonskii diagram

S0→S2 by absorption - internal conversion to S1 → either intersystem crossing to T1 or fluorescence or internal conversion to S0. If in T2 can do photochemistry or phosphoresce slowly to S0.

Intersystem crossing

Transfer from singlet to triplet multiplicity (spin flip)

Quantum yield

no. photoinduced events / no. photons absorbed

Fluorescence

Rigid molecule with no heavy atoms

Internal conversion

Flexible / rapidly rotating molecules

Intersystem crossing

Heavy atom molecule

Photosensitizer

Good chromophore forms triplet state when excited

DNA sequencing

First generation, second/next generation, third generation

First generation sequencing

500-1000bp, short read, Sanger, Maxam+Gilbert, Sanger chain termination

Next generation sequencing

50-500bp, short read, 454 pyrosequencing, Solexa, Ion torrent, Illumina

Third generation sequencing

Tens of Kb, long read, PacBio, Oxford nanopore

Primers on forward strand

Sense strand binds reverse primer at 3 prime end

Primer design

18-25 nucleotides, 40-60% GC base pair content, melting temp 55-65 degrees, absence of secondary structures

Maxam and Gilbert

Chemical modification adds radioactive label at 5’ end, purification and chemical treatment

Maxam and gilbert chemical treatments

Can cleave A+G, G, C+T, C by hot piperidine

What cleaves A+G in Maxam gilbert?

Formic acid (N7 protonation)

What cleaves G in Maxam gilbert?

Dimethyl sulfate (Methylated at N7)

What cleaves C+T in Maxam gilbert?

Hydrazine (C6)

What cleaves C in Maxam gilbert?

Hydrazine + salt (NaCl) (cleaves)

Limitations of Maxam gilbert

Complex, hazardous chemicals, laborious, radioactive labelling, low throughput

Sanger sequencing

Chain termination using ddNTPs

Limitations of Sanger

Can take years, costly

Sanger dye terminator

Uses fluorescently-labelled nucleotides, one pot, automation, parallel sequencing,

Advantages of sanger dye terminator

Single tube, no radioactivity, automation, capillary electrophoresis, direct readout

Limitations of sanger dye terminator

Dye peak issues, short read length, low throughput, higher cost, limited multiplexing capacity

Reversible terminator sequencing (illumina)

Unique fluorophores, 3’-o-azidomethyl blocking group to make temporary halt, then cleaved by TCEP

454 Pyrosequencing

Detects pyrophosphate release by its conversion to ATP that substrates for luciferin converts to oxyluciferin to emit light, sequential flow

Electrophoresis staining

SYBR used as less needed (safer) emits bright green light, Ethidium Bromide not used as cancer causing

Reagent for breaking disulfide bond (between Cys)

reducing agents DTT or BME

Alkylation of proteins to prevent reversibly forming disulfide bonds

Reagents iodoacetic acid, iodoacetemide, NEM

Acid hydrolysis

6N HCl, 120 degrees, 24h, destroys Trp, degrades Ser, Thr, Tyr, Asn→Asp, Gln→Glu

Base hydrolysis

2-4N NaOH, 100 degrees, 8h, Destroys Cys, Ser, Thr, Arg, Preserves Trp

Pre column derivatization

Ortho-phthaladehyde (OPA) or FMOC, using MPA

Post column derivatisation

Ninhydrin to make Ruhemann’s purple

IEC buffers low pH

Acetate, citrate, MES (cation exchange)

IEC buffers neutral pH

Phosphate, MOPS, HEPES

IEC buffers high pH

Tris-HCl (anion exchange)

Soft ionisation

ESI, MALDI

Protein sequencing (N terminal)

Sanger and Edman degradation

Sanger sequencing method reagent

dinitrofluorobenzene (DNFB)

Edman degradation reagent

phenyl isothiocyanate (PITC)

Endopeptidases (protein hydrolysis)

Trypsin, Chymotrypsin