Lesson 1 CD – Short answers
CD 1:1 a) rSM14-M20 is the most structured before denaturation. Motivation: in the left column, CD-spectra as a function of wavelength are shown. In the right column, ellipticity at a chosen wavelength (216 nm) as a function of temperature is shown. From the diagrams in the right-hand column we can see that the mutant with the highest magnitude of ellipticity at 25 oC is rSM14-M20, which indicates that this mutant also has the highest structure content. A minimum at this wavelength indicates b-structure. That this is the native form is shown by the reduction of ellipticity at this wavelength on denaturation. b) The denatured state has a larger contribution of random coil (200 nm) and tendencies towards helix formation (shoulder at 222 nm).
CD 1:2 c) rSM14-M20 also has the highest melting point (the midpoint of the denaturation curve, show the location in graph for complete answer) a) In this wavelength area (far-UV) the peptide group absorbs, and by studying the ellipticity (how the molecule ‘turns’ light, the difference between absorbance of right- and left-handed circularly polarized light) it is possible by CD to estimate the secondary structure content. At 222 nm the alpha helix shows a minimum, and the magnitude of this is proportional to the amount of helix. The helix content can thus be estimated from the magnitude of the signal. b) The amount of helix is in creased because the negative signal is increased.
(Note that these spectra are difference spectra where the complex minus its parts is shown!) c) For apo 1:1 and for holo 2:1 (peptide:CaM). Until these ratio are reached, the absolute value of the ellipticity is almost linearly increased, but after 1:1 and
2:1 ratios have been reached there is no more change in ellipticity. d) In this wavelength region (near-UV) aromatic residues absorb light (Trp, Tyr,
Phe), and significant ellipticity in this area indicates ordered side chains
(which thus turns polarized light in a preferential direction). e) In Tr1C both the ellipticity and the fine structure of the signal is more pronounced in the presence of peptide. This indicates that tryptophanes and/or phe/tyr become more ordered in the complex. This can occur either because Trp is present in the interaction surface, or because the protein core in general becomes more ordered in the complex.
CD 1:3 a) The native protein, which has a high propensity of helical structure (bimodal minimum, one of which is located at 222 nm), unfolds to a random conformation at 75 oC. At further elevated temperatures, b-structure
(minimum at 216 nm) is formed which is then maintained as the protein is cooled down to 25 oC. b) Perhaps the protein forms fibrils/plaques, which contain a large amount of bstructure? Perhaps these could aggregate and accumulate in the cancer cells? c) Only spectra for the wt are shown. It would be interesting to see how the mutations affect the protein structure as well as its fibrillation properties.