Biochemistry & Biophysics

• Δ

  • from carboxyl group

  • position of all double bonds is given

• ω

  • from methyl end

  • only position of first double bond is given

• by head group

  • phospholipids: head group with phosphate

  • glycolipids: sugar head group

  • sterols: steran

• by backbone

  • glycerolipids: glycerol

  • sphingolipids: sphingosine

  • sterols: steran

• fluidity: double bonds, methyl groups

• thickness: length of FAs -> influence on conductance

• curvature: ratio of head group and FAs -> different for vesicles and membranes

• surface charge: different head groups

• erucic acid 22:1 Δ13

  • 18:0 (stearic acid) produced in plastid

  • 18:1 (oleic acid) desaturated in plastid

  • two rounds of elongation in ER (?)

• ricinolic acid 12-OH-18:1 Δ9

  • 18:0 (stearic acid) produced in plastid

  • 18:1 (oleic acid) desaturated in plastid

  • oleate 12 hydroxylase introduces hydroxy group (where?)

• vernolic acid 12,13-epoxy-18:1 Δ9

  • 18:0 (stearic acid) produced in plastid

  • 18:1 (oleic acid) desaturated in plastid

  • Δ12 epoxygenase introduces epoxy group (where?)

Abb.

glyoxylate cycle:

• isocitrate —> glyoxylate + succinate (isocitrate lyase)

• glyoxylate + acetyl-CoA —> malate + CoA (malate synthase)

citrate cycle:

• isocitrate + NAD+ —> alpha-ketoglutarate + NADH + H+ + CO2 (isocitrate lyase)

• alpha-ketoglutarate + NAD+ + CoA —> succinyl-CoA + NADH + H+ + CO2 (isocitrate dehydrogenase)

• succinyl-CoA + GDP + Pi —> succinate + GTP + CoA (succinly-CoA synthase)

• succinate + FAD —> fumarate + FADH2 (succinate dehydrogenase)

• fumarate + H2O —> malate (fumarase)

• mevalonate pathway

• desaturation of FAs

• phospholipid synthesis

• sphingolipid synthesis

• TAG synthesis

• glyoxylate pathway

• isocitrate —> glyoxylate + succinate (isocitrate lyase)

• glyoxylate + acetyl-CoA —> malate + CoA (malate synthase)

• used to gain energy for growth from lipids (mainly germination of seeds)

• plant species: ?

• FA synthesis in plasmid

• FA exported to cytosol

• incorporated in glycolipid in Kennedy pathway (ER) ?

• DAG pathway: attached to choline/ethanolamine ?

• brought to plasmamembrane

Abb.

• synthesis

  • FA: FA synthesis (plastid)

  • choline: attached in DAG pathway (ER)

• degradation:

  • FA: beta oxidation (glyoxisome)

  • choline: ?

Abb.

• synthesis

  • FA: FA synthesis (plastid)

  • glycerol: attached?

• storage: ?

• degradation

  • lipase releases FA from glycerol

  • FA: beta oxidation (glyoxysome)

• long chain base (LCB): palmitoyl-CoA + serine

• cermade (Cer): LCB + FA

• glycosylceramides (GlcCer): Cer + sugar head (hexose)

• glycosylinositolphosphorylceramides (GIPC): Cer + phosphoinositol

  
  

• head group attachment specific to backbone -> signal for certain complex sphingolipid

• wound response

• root/flower development

• defense mechanism

• bacterial compund: coronatine (pseudomonas)

  • binds to JA-Ile receptor

• plant hormone: JA-Ile

• cutin

  • complex structure

  • below ground (root)

• suberin

  • layers

  • aerial surface

• hydroxylated FAs

• separates a mixture of dfferent compionents by properties

• separation by: boiling point, hydrophobicity, interaction, size, charge (physicochemical characteristics)

• utilizes stationary phase (containing the mixture) and a stationary phase (for separation)

• liquid or gas mobile phase possible

• analyte: substance to be separated (containg comppund of interest)

• eluent: mobile phase leaving the coloumn/chromatography after being separated

???

• transmethylation

  • removes FA from headgroup

  • ester bond of FA and methyl group

  • makes complex lipids volatile for analysis

• dimethyloxazoline (DMOX)

  • gives location of double bonds

• trimethylsinylation

  • H of funtional groups exchanged for TMS

  • makes polar compunds suitable for GC-MS

• O-phthalaldehyde (OPA)

• mobile phase: carrier gas, not reactive at high temperatures (He, Ne, Ar)

• stationary phase: long thin coloumns with silica layer inside

• advantages:

  • high resolution

  • retention time stability

  • reproduciable separation

• disadvantages:

  • analytes must be

    • volatile

    • relatively non-polar

    • thermostable

    • <600 Da

• FID (flame ionisation detection)

  • ions fromed by burning the compound in a hydrogen flame

  • detected via current measured

• MS (mass spectrometry)

  • ions formed b ydifferent methods

  • detected via m/z ratio

• compounds burned in hydrogen flame —> ions formed

• ions attracted to electrodes

• current measured proportional to amount of reduced Cs

• advantages

  • very sensitive

  • works for very organic analyte

  • accurate over seven orders of magnitude

• disadvantages

  • consumptive method

  • dependent on standards

  • not possible to get information on unknown compounds

• high performance liquid chromatography

  
  

• high chromatographic resolution —> sharp and high peaks

• high back pressure —> good pumps needed

• hydrophobic solvent —> mobile phase

• hydrophilic stationary phase

• normals phase HPLC: hydrophilic eluents

• reverse phase HPLC: hydrophobic analytes

1. ion source: generates ions in the gas phase from sample (ESI, MALDI, APCI)

2. mass analyser: separation of ions according to their m/z (Q, TOF, FT‐ICR, QqQ, Q TOF)

3. detector: detcetion (photomultiplier, MCP)

• EI (electron impact)

  • highly energetic electrons create ions

  • combined with GC

• ESI (electron spray ionisation)

  • high voltage creates charged droplets

  • combined with LC

    
    

• EI (electron impact)

  • hard ionisation technique

  • fragments created (charged and uncharged —> charge stays with one piece)

• ESI (electron spray ionisation)

  • soft ionisation technique

  • adducts created (sometimes only positive or only negative ESI possible)

  • in most cases no fragmentation

• TIC (total ion chromatogram/count/current)

  • sum of all intensities of all m/z signals at one time point

  • sum of I (for all m/z) vs t

• EIC (extracted ion chromatogram/count/current)

  • intensity of a specific m/z measured over time

  • I vs t for specific m/z

• mass spectrum

  • intensity of m/z of all compounds contained in one sample measured at one time

  • I vs m/z for specific t

• 3 quadrupoles (2 mass analyses, 1 collision cell) after ionisation

1. product ion scan

   • all fragment masses of one precursor

   • select, CID, scan

   • information:

2. precursor ion scan

   • single fragment of all precursors

   • scan, CID, select

   • information:

3. neutral loss scan

   • single neutral loss of all frgaments

   • scan, CID, scan m/z offset

   • information:

4. selected reaction monitoring (SRM)

   • single fragemnt of selected precursor

   • select, CID, select

   • information: reference table with precursors and fragments, check which lipids are in a mixture and their abundance (semiquantitative)

Abb.

• ESI

  • benefits:

    • soft ionisation technique

    • compunds of higher molecular weight can be analysed

  • limitations:

    • builds adduct

• TOF

  • benefits:

    • exact mass information (data base search for sum formula possible)

    • big range of mass

    • high sensitivity

    • high resolution

  • limitations:

    • complex lipids -> hard to identify individual compounds

    • expensive

    • detector can be saturated

• terpenoids

  • contain isoprene subunit

  • gibberilins, abscisic acid, strigolactons

• alcaloids

  • heterocyclic basic compounds

  • nicotin (real), mescalin (proto-), coniin (pseudo-)

• phenylpropanoids

  • from Tyr and Phe

  • lignans, lignin, cutin, suberin, coumarins

• mevalonate pathway (cytosol/ER)

  • pyruvate -> actyl CoA -> -> mevalonate -> -> -> isopentenyl pyrophosphate (IPP)

  • for synthesis of sterols, ubiquinones

• DOXP pathway (plastid)

  • pyruvate -> glycerinaldehyde-3-phosphate -> 1-deoxy-D-xylulose-5-phosphate (DOXP) -> 2-C-methylerythritol-4-phosphate (MEP) -> -> -> -> -> IPP

  • for synthesis of carotinoids, diterpenes

• cleavge of phsopahte groups

• allyl cation is formed

1. IPP synthesis -> mevalonate pathway (ER, cytosol) or DOXP pathway (plastid)

2. formation of terpene skeletons -> prenyl synthases

3. terpene modification

4. oxidative modfication

• heterogenous group

• mostly heterocyclic basic compounds

• derive from amino acids -> therefore N in ring or attached

• subgroups

  • real: nicotin

  • proto-: mescalin

  • pseudo-: coniin

• lignans: ?

• lignins: H-lignin

• volatile: benzaldehyde ?

?

• lignans: dimers

• lignins: high-order oligomers

• gibberillins

  • from geranylgeranyl diphospahte in DOXP pathway (plastid)

  • one synthase forms two rings, another one the other two -> ent-Kauren (plastid)

  • oxidative modifications: oxidases add carboxy- and hydroxy groups (ER)

• abscisic acid

  • from DOXP pathway (plastid): IPP -> C40

  • C40 (zeaxanthin, a carotinoid) -> xanthoxin (plastid)

  • synthesis of ABA (cytoplasm)

• strigolactons

  • from DOXP pathway (plastid): IPP-> C40

  • beta-carotin

• brassinosteroids

  • derive from squalene (C30)

  • cyclisation

  • oxidation of b-ring

  • modifications of the side chain

• ABA

  • plants: from DOXP pathway (C40)

  • fungi: from farnesyl diphosphate (start with C15)

• GA

  • different after ent-kauren

• auxin

  • agrobacterium has different pathway than plants

• hydroxylation (activation or inactivation)

• oxidation (activation or inactivation)

• conjugation (inactivation)

• plants: C40 from DOXP pathway

• fungi: start with C15 (farnesyl diphsophate)

parts of the cell wall

• primary walls

• middle lamella

• pectin-rich corners

components

• hemicelluloses

• glycans

• pectins

• proteins

functions

• forms scaffold

• shape

• cell development

• cell division

• glucose -> galactose: epimerase

• glucose -> mannose: epimerase

• glucose -> glucoronic acid: oxidation of OH at C6

• glucose? -> rhamnose: methylation

Abb.

reducing

• ring opening possible

• oxidation of sugar -> psoitive Fehling reaction

• free anomeric carbon

• semi-acetal

non-reducing

• no ring-opening possible

• negative Fehling reaction

• acetal

cellulose

• beta-1,4-D-glucan

• linear

• occurence:?

callose

• beta-1,3-D-glucan

• helix

• occurence:?

Xyloglycans

• cellulose backbone and C6 sugars attached

Glucuronoarabinoxylans

?

• cross-linking macromolecules of the cell wall -> tighter than glycans

• introduce charges and ionic bonds

• D-galacturonic acid/D-glucoronic acid (charge) chain and sugars attached

• functions

  • determine cell wall porosity

  • provide charged surfaces

  • modulate wall pH and ion balance

  • regulate cell-cell adhesion

• hydroxyproline-rich glycoproteins (HRGP)

  • extensin: structural integrity

• proline-rich proteins (PRP)

  • extensin: structural integrity

• glycine-rich proteins (GRP)

• arabinogalacton (AGP)

???

• expansins: loosen cell wall -> initiates cell wall stretching

• tranport proteins

• defensive proteins (kinases)

• agrobacterium has tumor inducing (Ti) plasmid

• transfers it tp host cell via a pilus

• ORF of T-DNA is changed to gene of interest

• T-DNA (between right and left border of Ti plasmid) is implemented into DNA of host cell

• additionally

  • marker for successful transformation with GOI-Ti-plasmid

  • marker for implementation of T-DNA in plant cell

• agrobacterium

  • + best established for stable transformation

  • + works for many plants and fungi

  • - random integration of of GOI (+ mainly in one locus)

• ballistic transformation (gene gun)

  • + best established method for tansient expression

  • + works for nearly all organisms

  • - radom integration

• homologous recombination

• - still difficult

• - for flowering plants only in chloroplasts

• + precise integration (often multiple copies)

• agrobacterium produces vir-proteins that lead to T-DNA integration into the plant cell

• T-DNA contains genes for auxin, cytokinin and opines (what are auxins and cytokinins for?)

• opines can only be used by agrobacerium

• plant cell produces “food” for agrobacterium but cannt use it itself

• protection against something

• examples

  • insects

  • viruses

  • herbicides

• synthesis of something -> to optimise production

• examples

  • improvement of yield, fruit ripening, sterility

  • nutritional value

  • produce biomass for chemical industry

• greenhouse

• field

• tissue culture

• bioreactors: glass reactors, ponds

• the more elaborate the more expensive

• depends on the end product (how expensive)

• mammalian cell culture can be infectd by viruses -> plant cell culture will not (also less allergic reactions)

• gene stacking: crossing two plant lines each contaiing one gene

• retransformation: adding one gene to a plant line that already contains the other

• unlinked tansgenes: adding both genes separately

• linked transgenes: adding both genes together

• split reading frame: ?

• operons: ?

• single step engineering (what is that?)

  • changes in product composition

  • introduce new product like antibodies

• multiple step engineering (?)

  • increase in yield

  • increase something already produced in the plant

• feedstocks?

• building blocks for chemical industry

  • replaces fossil fuel

  • might compete with food production

  • some FAs are toxic (chemical defense of the plant)

• calendic acid (18:3 (8E, 10E, 12E))

• alpha-eleosteraic acid (18:3 (9E, 11E, 13E))

• puinic acid (18:3 (9Z, 11E, 13Z))

• used as feedstock for paints

• rapeseed oil

  • - competes with food production

• camelina

  • + can easily be transformed for arabidopsis (similar genome)

• cremly?

  • + plastic production

• castor bean

  • - not fo food production

  • + seed oil has hydroxy-FAs -> good for plastic production

sensitivity

• UV/Vis: absorption

• fluorescence: good photomultiplier -> very sensitive

• both: compatible with most analytes and different separation techniques

B

• emission occurs at energies lower tha excitation

• Stokes shift

D

• all of the above

• strong bonds, sometimes covalent

• basic part of most fluorescent experiments

• allows to measure molecules which are not fluorescent

Source lamp

→ excitation monochromator

→ sample holder

→ emission monochromator

→ Detector

valence electrons

• HOMO/LUMO

decrease

• higher temperature -> more movement (rotatinal relaxation)

• increase in anisotropy -> decrease in polarisation

i) c = lambda * nu

ii) ? = 1/lambda

iii) E = h * nu

• absoprtion peak < fluorescent peak -> correct

• fluorescence peak > phosphorescence peak

  • should be smaller

  • triplet state at lower energy than singlet state

• transitions to higher energy states tha first excited state are not displayed in emission

• internal conversion from S2 to S1 -> excitation to S2 from S0, emission only from S1 to S0

Lambert Beers law

• peak of emission spectrum -> intensity plotted against concentration of Cl-

• F0/F vs. [Q]

• determine slope

• F0/F = 1 + kq * T0 * [Q]

• F0/F = T0/T

• dispersion: white light is split up into its components wavelengths

• each wavelength has a different refractive index -> fouses at different position along the optical axis

• rainbow fringes around objects and decreased resolution

resolution power:

x = 0.61 λn/NA

• Use a higher NA Objective (with immersion fluid), use a smaller wavelength

  
  

resolution power:

x = 0.61 λn/NA

• not moving with sample: not sample or coverslip

• has to be one of the conjugate planes of the sample: eyepiece, lamp field diaphragm, camera chip

• Wollaston prism I B

• Condenser iris B

• Eyepiece field iris A

• Specimen A

• Phase Plate B

• no reversal of actions of first prism

• shear and rotation of O- and E-ray are not reversed

• most light will pass through analyser

• imagevwill resemble standrard brightfield image

• pinhole aperture: cuts off other light

• rotating mirror: scan point source across sample

c.

• always: wavelength, objective NA

• additionally in confocal microscopy: pinhole diameter

• advantages:

  • better signal to noise ration (due to use of CCD cameras)

  • less belaching and phototoxicity

  • higher speed -> live cell imaging and movies

• disadvantages:

  • less axial resolution -> fixed pinhole

  • less flexibilty to adjust components depending on sample

a.

• under resolution of standard fluorophore

• blurred image (one big blob!)

b.

• should be able to analyse most fluorphores

• low probablitity of all fluorophores appearing

• different images of random combination of fluorophores -> need to be combined

c.

• scanning pixel by pixel, line by line -> confocal microscope

• emission and depletion laser at the same time

c.

• eficiency over time -> distance -> kinetics

• FCS also suitable

d.

• function of excitation

• formed by excitation wave reflected by glass

c.

• b: nothing to do witn Moire pattern

• d. true for SMLM

H = T + V = Σpi^2/2m + ΣV(ri)

H: total energy

T: kinetic energy

V: potential energy

p: momentum

E = h * ν

ν: frequency

p = h/λ

p: momentum

λ: wavelength

general:

ih- del ψ/del t = H^ ψ

-> ψ is an eigenfunction of the hamiltonian

time-independent:

H^ ψ = E ψ

-> the energy of the wave function is an eigenvalue

• precision with which certain pairs of physical properties can be known is limited

• the more accurate one, the more inaccuratethe other

• example: position and wavelength

• particle can only be in box -> potential energy is infinitively high everywhere else

• wave function in box

  • as many extrema as the quantum number n

  • crosses the x-axis n-1 times

E_n ~ n^2

• particle in a box -> box formed to a ring

E_n ~ n^2

• like a spring -> force is that of a spring, potential energy the integral

E_n ~ n

• takes into account that bonds can break and compression is limited

• energy states get closer the higher the quantum number

• electrons in atom

• in classical atom model the electrons would fall into the nucleus

E_n ~ 1/n^2

<x> = ∫Ψ*(x) x^ Ψ(x) dx

• after system is disturbed

• describes probability of transition from one energy eigenstate to another

• defined by transition moment squred proportional to density of states

• stimulated absorption leads to transitions between spin states

  • W_i->f = B_fi * ρ

• same is true for stimulated emission (opposite transition)

  • W_f->i = B_fi * ρ

• this way there would be no Boltzmann distribution of energy -> there is also spontaneous emission (creaes Boltzmann distribution)

  • Wi->f = A_fi

A = log(I0/I) = c*d*ε

A: absorbance

I: intensity

c: concentration

d: thickness of cuvette

ε: molar extinction coefficient

assumptions:

1. electrtronic transitions are fast -> nuclei move after transition

2. transition arrows are orthogonal to R-axis (x-axis)

3. transition moment depends only weakly on nuclear coordinates and /or they do not change much in the transition

• eletronic transition from one vibratona energy level to another is more likely if the two vibrational wave functions overlap