Chapter 1 and 2 - CHM136

Infrared spectroscopy

• determines the functional groups of a molecule

• cm-1 - wavenumber

  • energy is proportional to 1/wavelength of the oscillation

• bonds in a molecule absorb photons of light from the IR region of the spectrum , causes change in bond vibrations

• bond in diff fg’s vibrate at different characteristics therefore the wavenumber of the absorbed light reveals the molecules fg’s

bonds

• in molecules are constanlty vibratins around a certain equilibirum distance - bond length

Molecular vibrations

• constantly stretching or bending motions

• s-1

Stretching modes of vibration

• vibration either lengthen or shorten bonds to distances slightly longer or shorter than their most stable bond lengths

• relative to the equilibrium bond length - constanlty gets shorter or longer or to the equillibrium

Bending modes of vibrations

• increase and decrease the angles between atoms relative to the equillibirum bond angles in the molecule

• each vibration occurs at slightly different frequency s-1

which molecuels undergo more stretching and bending vibrations

• molecules w/ more atoms

Number of vibrations

3n-6

Vibrating bodns create change in dipoles

• creates oscillating electric field

• these functional groups of the elctric field absorbs light between 4000 and 600 cm-1

IR spectroscopy

• when IR passes through a sample of th ecompound the photons w/ frequencies that match those of molecular vibrations are absorbed

  • those not absorbed read a detector and are converted to spectrum

IR spectrum

Hydrogen region

• 3600-2700 cm-1

• OH , NH, CH (sp3, sp2, sp)

• bonds with hydrogen vibrate at largest frequencies - due to lightness of h atoms

• terminal alkyne most high

• aldehydes ahve two medium peaks

Amines or amides

• narrower than alcohol peaks and broader than alkyne

• primary amines and amides produce 2 peaks

• secondary amines and amides produce only 1 peak

Carboxylic acid peaks

Triple bond region

• alkynes and nitriles (more intense)

Terminal alkyne

• induces a greater change in dipole therefore producing stronger signal

Finger print region

1500 - 500 cm-1

Spin

• all nuclei posses this quantum mechanic property

• spin state number is determined by atomic number and mass

H1 and c13

• only 2 possible spin states

• +1/2 or -1/2

Spins of 0

• nuclei w/ even atomic mass and numbers

odd nuclei

• can occupy several spin states

nuceli responding to magnetic fields

• yield significant information about the structure of the molecule containing the nuclei

• magenetic moments of nuclei in an external magnetic field (B0) align either parallel to the external field (alpha lower energy) or antiparallelt to it (beta, higher energy)

• alpha more stable because lower energy

delta E

• difference in energy between alpha and beta states - is small

• depends on the magnetic field

• stronger magnetic field results in larger delta E

Spin flip

• transition from nuclei between apha and beta states

Resonance frequency

• frequency at which a spin flip occurs

• this happens when EM radiation of a frequency corresponding to deltaE - to the nuclei - the alpha state are excited and move to higher energy beta state

• while the beta state moves to the alpha state

• this equalizes the population of a and beta states and produces detectable magnetic field that contains a frequency component for each hydrogen type

Free induction decay

• after excitation - the magnetic field weakens as the systme relaxes and goes back to equilibirum

• signal detection provides info on teh type of each hydrogen

• different chemical envrionments in a molecule cause variations int he magnetic field around the nuclei

H2 resonance

• CDcl3 - d is deuterium h2

• tuned to select a type of nucleus

• signals corresponding to the sample are much stronger than those of the solvent

TMS

• used as ppm 0

Integration

• the calculation of the area under the peak - that states how many hydrogens are present in this chemical environment

Chemical shift

Lenz’s law

• the principle that an induced current always generates a magnetic field opposing the applied field is knonwn as Lenz’s law

Local magnetic fields sigma B0

• when exposed to an external magnetic firled B0 electrons in a compound circulate creating small currents that generare local magnetic fields that oppose the external field

shielding

• the reduction in the magnetic field strength experienced by nucleus due to opposing magnetic fields of nearby electrons

• nuclei that are shielded by their envrionment - have electrons surrounding them produce signals toward the right

Upfield

• shielded nuclei by the envrionment that is surrounded by electrons produce a signal toward the right of the ppm spectrum

Downfield

• hydrogens that are less deshielded by surrounding electrons - hydrogens near electronegative atoms - produce signal that are downfield toward the left

TMS

• used as the reference compound

• has highly deshielded electrons that are chemically equivalent

Electronegative atoms

additive effect

• the more electronegative atoms the more deshielded the hydrogens are

Deshielding of functional groups

Carbon electronegativity

• isobutane more electronegative than propane and ethane

• as carbon is more elctronegative than hydrogen

RAG

• enzyme responsible for recombination

V gene segments are joined by recombination - forward

• joining segments have the same transcriptional orientation - most common

• the intervening DNA is loooped out and lost fromt he chromosome

• there is formation of an excision signal(signal joint) and a coding joint

V geme segments are joined by recombination - revers

When segments are initially oriented in opposite transcriptional directions, alignment of the RSSs requires a coiled configuration. Thus, joining the ends of the two heptamer sequences now results in the inversion and integration of the intervening DNA into a new position on the chromosome.

why is there a 12/23 base pair rule?

• may result from the asymmetric binding of RSSs to the RAG 1 and RAG 3 dimmer

• there are two identical rag-1 and 2 identical rag-2

  • favours the 12bop - because shorter - proximity

where are rag genes expressed?

• only in developin B and T cells

• RAG knockout mice fail to develop B or T lymphocytes, and RAG1 or RAG2 mutations are a cause of SCID (patients lack B and T lymphocytes).

Enzymatic steps in the Rag-dependent V(D)J rearrangement

1. Binding of the rag-1 and rag-2 proteins as well as a protein called high mobility group.

2. these bind to one of the rss’s that flank the coding sequences come together

3. Cleavage step - the endocnuleases of RAG makes single-strand cuts in teh DNA backbone preciseling between each coding segment and its RSS.

4. At each cutting point this creates a 3′-OH group, which then reacts with a phosphodiester bond on the opposite DNA strand to generate a hairpin leaving a blunt double- strand break at the end of the RSS.

5. 1 hair

6. 1 blunt end - This is the singal joint end : Ku70:Ku80 binds the DNA ends - and joined together bu a ligase protein - to make a loop and to be the signal joiont which will get deleted later but stray blunt - the - precise

Coding joints - what happens - imprescise coding joint

1. KU70 and KU80 proteins need to add some nucleotides to what was cut previoously.

2. done by Artemis/DNA-PK endonuclease activity opens the hairpin , modifies the ends of the hairpin by adding nucleotides .

3. modified by terminal deoxynucleotidyl transferase (TdT; pink). TDT adds random extra nucleotides (N nucleotides; red)

   V sequence end

   A-T-C-A-G-T-A-C-T-G-C T-A-G-T

   nicked hairpin

   The two coding ends (e.g., V and J) are ligated by the DNA ligase IV/XRCC4 complex

P and N nucleotides in the VJ junction

• N nucleotides are added by the enzyme terminal deoxyribonuclotuidyl transferase (Tdt)

  • Tdt only expressedn in developing b cells

• No P Or N nucleotides int he signal junction

• these help ensure the coding joint is properly ligated

and improves diversity of the coding joint

P nucleotides

• palindromic nucleotides - mathces the sequence

• due to the asymmetric nucking of a hairpin end

• random residues

• involved in diversity of the coding joint

N nucleotides

• non templated nucleotides

D-J heavy chain rearrangement

• same thing happens for heavy chain v-d and light chain v—j rearrangement

1. DNA hairpin at coding ends generated by RAG (heptamers are ligated to form the signal joint

2. Artemis:DNA -PK complex opens DNA hairpins generating palindromic P-nucleotides

3. N-nucleotide additions by Tdt

4. Pairing of strands

5. unpaired get removed by an exonuclease

6. DNA ligase addition of nucleotides for the gaps and then ligated together form coding joint

what contributes to the third hypervariable region

• the variable addition and subtraction of nucleotides at the juncrions between gene segments

• but leads to recombinational inaccuracies

• Increased variability of CDR3

  2/3 of rearrangements are non-productive (out of frame) this leads to in-frame stop codons.... TAA, TAG or TGA in J or C region
  A trade-off between greater diversity and loss of efficiency

• some can also be produyctive

• because of recombination even if the v, d and j genes are the same they can produce different antigen binding sites because of this

IgM and IgD

• derived from the same pre-mRNA TRANSCRIPT AND are both expressed oin the surface of mature b cells and act as receptors

• b cell does not need to rearrange its genes a second time to make IgD

• only one rearrangement for both - and express both of them

• it is dependent ont he processing

Transmembrane and secreted forms of immunoglobulins are derived from the same heavy-chain sequence by alternative RNA processing

• two poly AAA sites

• either the secretion or the tranmembrane process is spliced out

• different forms of rna processing takes place results in different immunoglobin forms