Pull down Assay
This technique was used to determine a physical interaction between two or more different proteins: Protein 1 is immobilised to a purification column, and sample containing another protein of interest (protein 2) is passed through the column. This allows for the determination of whether protein 1 and protein 2 physically interact through analysis of the elutant. This was carried out to study some of the interactions of cwc2 with other protein components in the spliceosome.
Limited proteolysis
Tryptic peptide mass fingerprinting was used to obtain Cwc21-240 stable fragment. Size-exclusion chromatography was the main technique used to purify this fragment.
Figure 1. Separation of molecules by size due to differential entry into beads in column matrix
Cloning, expression and mutagenesis
The cwc2 gene and its deletion mutants coding for its RRM (Cwc2124-234) was expressed with an N-terminal hexahistidine tag and cleaved with a tobacco etch virus protease. Point mutations were engineered using the QuickChange Site-Directed Mutagenesis strategy (Stratagene).
Figure 2. Mutagenesis protocol using primers in QuickChange
Crystallographic procedures
Crystallisation was carried out using the sitting-drop vapour-diffusion method, by mixing 2 μl of protein solution with 1 μl reservoir solution of 30% PEG 5000 monomethyl ether. Cryoprotection with glycerol content was used and a highest resolution of 2.0 Å was obtained.
Figure 3. Diagram depicting methodology of crystallisation via a sitting drop
Analysis of the structure
Software analysis of structures was done using the DALI server. Structural superimpositions to look at alignment of domains were carried out with the SSM tool Furthermore, secondary structure elements of scCwc21-227were extracted with PROMOTIF.
Electrophoretic mobility shift assay
[32P]-5’-labeled, in vitro transcribed yeast U6 snRNA was incubated with recombinant Cwc2 full-length protein or fragments depending on whether whole protein or only domain interactions were being studied. RNA and RNA-protein complexes were resolved on a native polyacrylamide gel.
This assay was used to study the above protein-RNA interactions, where a radio-labelled RNA strand is incubated with the specific RNA-binding fragment. The migration of the unbound RNA strand through a non-denaturing polyacrylamide gel electrophoresis. The bound protein retards the mobility of the RNA strand in comparison to the unbound RNA strand.
Figure 4. Separation of nucleic acid and protein in complex and unbound nucleic acid to determine binding and sequence bound
Finding if a stable and defined complex could be reconstituted between Cwc2 and U6 snRNA in solution:
1) full-length Cwc2 was incubated with U6 snRNA at a molar ratio of 10:1 and then subjected to size-exclusion. The elution profile was dominated by a single peak that corresponded to a monodiperse complex with the molecular weight of 260 kDa and the elution volume of the complex was in agreement with the ones of the individual components
2) Subjected the mixture to UV irradiation and the complex was then hydrolysed with nucleases and endoproteinases, and titanium dioxide enrichment was subsequently performed
3) Crosslinked peptides were identified by the corresponding fragments obtained after higher energy collision-induced dissociation (HCD)
4) The composition of the crosslinked RNA moiety was calculated from the difference between the mass of the crosslinked species and the calculated peptide mass
2) Subjected the mixture to UV irradiation and the complex was then hydrolysed with nucleases and endoproteinases, and titanium dioxide enrichment was subsequently performed
3) Crosslinked peptides were identified by the corresponding fragments obtained after higher energy collision-induced dissociation (HCD)
4) The composition of the crosslinked RNA moiety was calculated from the difference between the mass of the crosslinked species and the calculated peptide mass
The UV-Irradiation method:
· UV-induced cross-linking is used to investigate protein-DNA/RNA. It utilises the natural reactivity of the nucleic acid bases after irradiation at a wavelength of 254 nm. Excitation by UV light causes subsequent radical-based reactions, which lead to the formation of a covalent bond between the nucleic-acid base and an amino-acid residue, (a.k.a a zero-length cross-link). C, K, M, F, W and Y residues are the most reactive towards cross-linking; however, all of the common amino acids except proline can form cross-links (Shetlar et al, 1984)
· These excited states of the nucleic-acid base have a short half-life, thus cross-links can only be formed to amino acids that are in contact or in close spatial proximity to the excited nucleotide (Budowsky et al, 1986). This type of cross-linking leaves the three-dimensional structure of the protein–RNA complex mostly unaffected.
· Mass spectrometry (MS)(See figure S7) is preferred for the unbiased identification of the cross-linking sites and thus of the individual cross-linked protein residues. UV cross-linking combined with MS can be used as a powerful tool in the identification and characterisation of protein–RNA contact sites.
 |
S7 Product ion spectrum (fragment spectrum) of CWC2 peptide FVSPFALQPQLHSGK (F47-K61) cross-linked to U with loss of H2O. y and b-type fragment ions that unambiguously reveal the sequence of the cross-linked peptide are assigned. The a2- and b2- ions show a mass shift of 94 a.m.u. that is indicative of cross-linked U –H2O (Kramer et al, 2011) and importantly, the immonium ion (IM) of F also exhibits an additional mass of 94 a.m.u., thus strongly suggesting that F47 is the actual cross-linked amino acid (all 94 Da mass shifts in red). Identified peptide fragments, peptide sequence and cross-linked nucleotides are indicated at the upper right. The insert shows the precursor ion spectrum. Asterisk denotes neutral loss of ammonium. a.m.u. = atomic mass units.
|
