Advanced Biochemistry. Summary

Exam Revision, 2014

21 Pages, Grade: 1,0



Advanced Biochemistry

1. Wahl - Splicing

Explain why pre-mRNA splicing is an energy-neutral process. The process still requires energy in the form of NTP-hydrolysis - why and how are these consumed?

- Splicing consists of two energy-neutral trans-esterification reactions
- ATP (mostly, for DEXD/H-box proteins) and GTP (for Snu) are required for rearrangements

Consensus sequences of splice sites and branch points are short and degenerate. What are the advantages of them being so? How does the spliceosome control the choice of correct splice sites and branch points?

- Advantage: Flexibilty of splice sites due to weak interactions ^ alternative splicing
- Splice site fidelity: Repeatedly recognized by different factors, multiple weak interactions sum up (binding synergy)

You've got two cell extracts; one with wtPrp2, the other with a temperature sensitive mutant that supports splicing at 30°C but inhibits it at 37°C.

How can you check on which splicing step the Prp2 mutant interferes? Basis/background of your approach? Experimental setup? Control? Detection method?

- Conduct in vitro splicing experiment with MS2-tagged pre-mRNA and cell extract containing either wtPrp2 (control) or mutant @37°C
- MS2-MBP pulldown
- Dent.PAGE& silverstaining
- Compare bands to size of U snRNPs and mRNA intermediates for information on complex composition

What are the differences between the ribosome and the spliceosome?

- Spliceosome is assembled de novo for each intron
- Vectorial process
- Extensive compositional and conformational rearrangements
- Transesterfication reaction
- Splice site flexibility: not every intron is spliced, but every codon is read out

2. Heyd - Alternative Splicing

What are „exonic splicing enhancers"and „exonic splicing silencer"?

- Cis-acting elements within the exons of the pre-mRNA that regulate alternative splicing
- Recruit SR-proteins (ESE) or hnRNPs (ESS)

What are cis- and trans-acting elements that support or inhibit exon inclusion?

Abbildung in dieser Leseprobe nicht enthalten.

How can you detect cis- and trans-acting elements experimentally?

- Detectingcis-actingelements: Minigenes
- Construct minigene that is alternatively spliced under known conditions (e.g. activation with PMA)
- RT-PCR & gel analysis of both conditions
- Mutate/truncate different nucleotides/regions to identify the position of cis-acting element (^ no more alternative splicing under second condition)

- Detecting trans-acting factors: RNA-protein-interaction studies
-Incubate biotinylated RNA containing cis-acting element with protein, pulldown

Large-scale analysis: splicing sensitive microarrays (Exon Array), Deep Sequencing (RNASeq)

Validation: RT-PCR followed by qPCR with radioactive/4primers or isoformspecific primers


3. Freund - NMR

How are peptides processed in late endosomes? / Describe the process exchanging self peptides with pathogenic peptides on MHCIIs in the late endosome.

- Endocytosed antigenes are degraded into peptides in endosomes
- CLIP blocks MHCII peptide binding pocket (stabilises MHCII)
- HLA-DM catalyses exchange of CLIP and peptide by stabilising open conformation of MHCII
- Loaded MHCII travels to cell surface

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How can Small Molecules interfere with peptide loading onto the МНС II?

- Small molecules are able to catalyse MHCII CLIP-peptide exchange
- Bind to peptide binding pocket, keeping it in an open conformation

What is an Adverse Drug Reaction?

- Harm associated with the use of given medications at a normal dosage during normal use
- Some molecules can bind to peptide binding pocket, altering the peptide repertoire and thereby inducing hypersensitivity reaction:

Abacavir can bind inside the peptide-binding groove of HLA-B[*] 57:01, thereby enabling the presentation of peptide ligands that normally cannot bind in substantial amounts. Because T cells are generally tolerant only to МНС-restricted peptide ligands presented during T-cell development in the thymus, presentation of an altered repertoire of class I MHC binding peptides will be perceived as being foreign and trigger CD8+ T-cell responses.

What happens to MHC II complexes when no foreign peptide is around?

- MHCII can be loaded with self-peptides derived from cytosolic self-proteins by autophagy

What is the principle of detecting and assigning proteins and peptides in NMR? / What is the procedure in NMR spectroscopy for resonance assignment of peptides and proteins?

- Certain atomic nuclei possess as spin and a magnetic moment (1H, 15N, ...)
- The chemical environment (neighbouring atoms) influences their resonance frequency
- An external magnetic field is applied
- Radio frequency pulses excite the spins
- Magnetic resonance response is obtained (FID)
- Fouriertransformation
- Interpretationofspectrum

You add a proline-rich sequence to a purified 15N labeled SH3 domain and record a HSQC spectrum afterwards (spectrum below the question). Describe the aminoacid-spots, name the X- and Y-axis. Now you add a SH3 binding domain with 100 mM in stepwise higher concentrations. What will you see in the spectrum compared to the previous one?

- OR SH3 binding domain: Shifts of aminoacids involved in the binding site due to changed chemical environment

Explain the differences in ligand- and protein-observed NMR-setups. / Describe the difference between ligand- and protein-observation in detection of ligand-protein interactions (NMR)? (same as the first test)

- Ligand: small amount of protein needed, no labelling of protein, suitable for proteins of any size, information about binding-epitop of ligand, suitable for HTP, no or little information about interaction site of protein, lD-spectrum is sufficient
- Protein-observed: large amount of protein, protein has to be labeled, more dimensional spectrum (HSQC), more effort, limited size, information about interaction site possible

A MHCII protein (lsN-labeled) interacts with a low affinity peptide (unlabeled). Which NMR-method would you assign? How does the spectrum look like (draw) if 100% complex is built? (Label axises) What happens when a high affinity ligand is added over time? What happens if HLA-DM is added?

- NMR-method: record HSQC spectrum, because MHCII is too big for 1D-spectrum. Can label both subunits individually and superimpose both spectra
- X-axis: 1H (ppm), Y-axis: 1SN (ppm)
- If high affinity ligand is added —> first ligand will be replaced by high affinity ligand (then chemical environment and thus chemical shift changes and ligand signals appear as distinct signals from protein)
- HLA-DM catalyzes exchange—> new complex is built faster

Abbildung in dieser Leseprobe nicht enthalten.

4. Heinemann - IntracellularTrafficking and Sorting

How are misfolded proteins detected by the ER and degraded?

- Recognition of misfolded proteins by glycosylation state (UGGT)
- Retention factors (Hsp70-type and glycan-dependent chaperones) prevent exit
- Lectin chaperones (calnexin, calreticulin) try to remodel protein
- Eventually degradation by ERAD-system
- Dislocation into the cytosol
- polyubiquitination by ubiquitin ligases at ER membrane
- AAA+ ATPase removes protein from membrane
- proteosomal degradation

Outline the principle of intracellular transport in eukaryotes. Are there species-specific differences?

- Transport by diffusion
- Active transport by motor proteins (ATP hydrolysis)
- Conserved between yeast and mammals

Draw a basic scheme of vesicle transport in eukaryotic systems.

Intracellular Transport

Abbildung in dieser Leseprobe nicht enthalten.

- Initiation of coat assembly: recruition of cargo proteins and SNAREs
- Budding: coat proteins form mesh-like structure, membrane curvature increases
- Scission: vesicle is released by dynamin
- Uncoating: coat disassembly by GTP or PI hydrolysis or uncoating enzymes
- Tethering: vesicle tethers target membrane by GTP-Rab and tethering factors
- Docking: v- and t-SNAREs form four-helix bundle
- Fusion: SNARE-complex promotes fusion ofvesicle and target membrane


You have cloned a gene that may encode a new dynamin protein. How would you prove this? / You have cloned a gene and believe it belongs to the dynamin superfamily. How would you prove this biochemically? (3 Answers) (same as the first test)

- Membrane tubulation assay (Beobachtung von Liposomen-Tubulation mit EM)
- Oligomerization assay (e.g. ultracentrifugation, SEC)
- Endocytosis assay
- Crystallisation
- Sequence alignments
- Stimulation of GTPase activity by providing PIP2 tubules
- Differential interference contrast microscopy (DIC)

Name 3 members of the dynamin superfamily and describe their function.

- Dynamin 1: Vesicle scission (e.g. endocytosis, budding of clathrin-coated vesicles)
- MxA: viral resistance (confer resistance against RNA viruses, interacts with viral RNPs)
- Mitofusinl & OPA1: reorganization of mitochondria

Abbildung in dieser Leseprobe nicht enthalten

Abbildung in dieser Leseprobe nicht enthalten

non-hydrolysable GTP analogues:

Abbildung in dieser Leseprobe nicht enthalten

G-Protein families

- Heterotrimeric G-proteins in 7TMR-signaling: Transducin
- Initiation, elongation and termination factors in protein synthesis
- Ras-like GTPases in signal transduction: Ras, Rab, Rho, Ran, Rab, Arf, Ari, Sar
- Dynamin superfamily in remodelling of membranes


[*] SH3 domain binds to proline-rich sequences ^ formation of complex
- Describe aminoacid-spots?
- X-axis: 1H (ppm), Y-axis: 15N (ppm)
- Stepwise??
- Unlabeled SH3 domain: Shifts, because of competitive binding. Free SH3 domain will sequester proline-rich sequences ^ unbound 15N labeled SH3 domain

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Advanced Biochemistry. Summary
Free University of Berlin  (Institut für Chemie und Biochemie)
Advanced Biochemistry
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