Q.1. What is the packing ratio (stoichiometry) of Histones in a nucleosome core particle?

A. H2A₂H2B₂H3₂H4₂

Q.2. The alpha subunit of Na-channels contains more than 2,000 amino acids. How many nucleosomes (approx.) would be needed to pack the gene coding for this protein into the chromosomal structure?

A. each nucleosome contains about 200bp of DNA plus 100-150bp of linker DNA between the next nucleosome: app. 18 - 20 nucleosomes

Q. 3. What type of amino acid residues are involved in nucleosome-nucleosome interaction? Which post-translational modification affects this interaction? What structural feature in the nucleosome crystal structure explains why histone-DNA interaction is not DNA sequence specific?

A. Arg and Lys (at N-terminal H3 and H4 residues) are involved; modified by acetylation (removes + charges); contact sites show interaction between protein backbone atoms and arginine residues with minor groove surfaces (note: only R residues are involved)

Q. 4. Keratin is part of which cellular structure? What is its supramolecular structure in mammalian hair cells?

A. intermediate filament of cytoskeleton; The protofilament is an association of keratin dimers and protofilaments assemble into microfilaments. Fig. 7-25 in Voet&Voet shows the organization of the macroscopic filament formation in hair

Q. 5. Macromolecular structures in cells can be shown to have self-assembly properties in vitro. In vivo, however, the assembly of those structures is often assisted by 'helper proteins'. What are these proteins called and give an example (for nucleosome assembly, for example).

A. these proteins are called chaperones; nucleoplasmin

Q. 6. What is the mechanism of subunit shuffling in cytoskeletal fibers? What is the energy source of this shuffling process (distinguish different filament types for different energy sources)?

A. treadmilling; nucleotide hydrolysis: ATP in actin (microfilaments), GTP in tubulin (microtubules)

Q. 7. What is the importance of having regions of high variability, like the CDR, on antibodies? On T-cell receptors?

A. antibody CDR (complementarity determining region) interact with antigen epitope (surface), the variability provides many different binding epitopes on a class of proteins with similar tertiary and quaternary structure; T-cell receptors recognize MHC class I with bound peptide with their variable domain structure

Q. 8. How can he X-ray structure of the immunoglobulin fold help us understand that these regions of high variability don't affect the stability of the variable domain (compare this problem to the non related structures found in the chymotrypsin superfamily of serine proteases)?

A. The globin fold in the variable domain is not variable as backbone beta strand conformations from different antibodies can be superimposed; only the loops connecting the beta-strands contain variable sequence and provide a microheterogeneity in their surface conformation (se also problem 7); families of serine proteases show distinct tertiary fold, but have a conserved catalytic site structure, here the active center is conserved, but not the overall structure, while in immunoglobulins the overall structure is conserved, but not the active binding site

Q. 9. What biochemical 'trick' was needed to solve the X-ray structure of immunoglobulins?

A. to proteolytically cleave it at the hinge region into two Fab and one Fc fragments (the Fc fragment contains a N-linked oligosaccharide unit)

Q. 10. Which of the following protein(s) is not a membrane protein: class I MHC, T-cell receptor, IgG, IgM, muscarinic Acetylcholine Receptor, Myoglobin?

A. IgG, Myoglobin

Q. 11. What are the major core constituents of N-linked oligosaccharides?

A. NAG (N-acetylglucosamine) and Mannose

Q. 12. You study a new plasma membrane protein and try to identify its topology. You already predicted 7 potential transmembrane spanning segments. Based on the sequence analysis, how could you predict the inside-to-outside orientation of the protein, i.e., if the N-terminal domain is extra- or intracellular? What is the superfamily of proteins we are dealing with called?

A. you can predict putative N-linked glycosylation sites; G-protein coupled receptors or 7-transmembrane helix proteins

Q.13. What is the mechanism of lectin binding to other proteins? Can it bind any protein?

A. lectins recognize oligosaccharides and do not bind do non-glycoproteins

Q. 14. Cadherins are one type of protein family involved in cell-cell interaction. Explain all potential analogies to the PDGF receptor based on their domain structure.

A. cytoplasmic domain, one single transmembrane segment (helical), large extracellular multidomain structure (contains 5 immunoglobulin fold like domains); forms dimer as active structure and prior to cadherin zipper formation

Q. The movement of eukaryotic and prokaryotic flagella are based on different mechanisms. What is the energy source of each process?

A. eukaryotic flagella - ATP; bacterial flagella - H+ gradient

Q. 16. How does a cell recognize if a protein has to be secreted or put into the plasma membrane?

A. the signal recognition particle (SRP) recognizes a the signal sequence (or signal peptide) at the N-terminal end of the newly synthesized (nascent) protein emerging from the ribosome

Q. 17. How can a protease inhibitor be a potential drug suppressing the proliferation of HIV but not affect the host physiology?

A. the inhibitor has much higher affinity for the viral protein than the host homologue

Q. 18. What is the structural principle of a leucine zipper; what general feature of an alpha helix does it follow?

A. leucine zippers are coiled-coil alpha-helix dimers stabilized by hydrophobic interaction with evenly spaced non-polar residues (one type is a leucine residue); the binding motif is the heptad sequence a-b-c-d-e-f-g with position 'a' being non-polar and position 'd' being a leucine residue only; all other positions are polar/charged

Q. 19. Explain the functional differences and similarities of Zn⁺⁺ ions in carbonic anhydrase and Zn-finger proteins.

A. Zn++ in carbonic anhydrase is tightly bound to active center and participate in the reaction; Zn++ in zinc finger motifs stabilizes the structure, but is not involved directly in DNA binding

Q. 20. Describe one of the motifs in the binding site of DNA binding proteins (interaction to major groove only).

A. choose form: leucine zipper; helix-turn-helix motif; zinc-finger; all motifs contain a short alpha helical structure that interacts with the DNA at the major groove surface; transcription factors always act as dimers