1 Supporting Information Salt-dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum Julie N. Reitter, Christopher E. Cousin, Michael C. Nicastri, Mario V. Jaramillo and Kenneth V. Mills* College of the Holy Cross, Department of Chemistry, Worcester, MA 01610 Figure S1. Diagram of the chemical steps of protein splicing. Figure S2. Sequence alignment of Hsa and Mma PolII inteins. Figure S3. Sequence of MIHHsaWT fusion protein. Figure S4. Temperature dependence of splicing. Figure S5. Comparison of protein splicing of MIHHsa fusion proteins. Figure S6. Time dependence of protein splicing and N-terminal cleavage. Figure S7. Influence of ion concentration and identity on splicing and cleavage reactions. Figure S8. Reductant-dependent mobility shift of precursor protein and Cys mutants. Figure S9. pH dependence of protein splicing. Table S1. MALDI-TOF/MS analysis of protein splicing and cleavage of MIHHsaWT, MIHHsaQN and MIHHsaQACA. Table S2. N-terminal sequencing analysis of protein splicing of MIHHsaWT. Experimental Methods References for Supplemental Information
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Supporting Information
Salt-dependent Conditional Protein Splicing of an Intein from
Halobacterium salinarum
Julie N. Reitter, Christopher E. Cousin, Michael C. Nicastri, Mario V. Jaramillo and
Kenneth V. Mills*
College of the Holy Cross, Department of Chemistry, Worcester, MA 01610
Figure S1. Diagram of the chemical steps of protein splicing.
Figure S2. Sequence alignment of Hsa and Mma PolII inteins.
Figure S3. Sequence of MIHHsaWT fusion protein.
Figure S4. Temperature dependence of splicing.
Figure S5. Comparison of protein splicing of MIHHsa fusion proteins.
Figure S6. Time dependence of protein splicing and N-terminal cleavage.
Figure S7. Influence of ion concentration and identity on splicing and cleavage reactions.
Figure S8. Reductant-dependent mobility shift of precursor protein and Cys mutants.
Figure S9. pH dependence of protein splicing.
Table S1. MALDI-TOF/MS analysis of protein splicing and cleavage of MIHHsaWT,
MIHHsaQN and MIHHsaQACA.
Table S2. N-terminal sequencing analysis of protein splicing of MIHHsaWT.
Experimental Methods
References for Supplemental Information
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Figure S1. Diagram of the chemical steps of protein splicing. Protein splicing is a four-step
process: (1) an amide-thioester rearrangement of the peptide bond linking the N-extein and
intein, (2) transesterification resulting in transfer of the N-extein from the side chain of the first
residue of the intein to the first residue of the C-extein, (3) cyclization of the intein’s C-terminal
Asn coupled to peptide bond cleavage, and (4) conversion of the thioester linking the exteins to
an amide and potential hydrolysis of the C-terminal aminosuccinimide of the intein to Asn or
iso-Asn. Inteins with C-terminal Gln likely proceed via a similar mechanism involving Gln
cyclization.
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Figure S2. Sequence alignment of Hsa and Mma PolII inteins. Alignment of intein sequences
by the EMBOSS Water sequence alignment tool.1 Conserved intein sequence motifs are
highlighted in green (Both inteins lack a central homing endonuclease domain, which would
comprise blocks C, D, and E.) Sequence identity shown as a bar between the two sequences,
sequence similarity shown with a dot. The gap in the Mma PolII intein sequence is the location
of the putative loop region in the Hsa PolII intein noted in the article.
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Figure S3. Sequence of MIHHsaWT fusion protein. The sequence of the N-extein is given in
red (maltose binding protein and linker) and green (native N-extein residues), followed by the
intein in blue, native C-extein residues in green, and the linker and poly-His tag in red.
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Figure S4. Temperature dependence of splicing. SDS-PAGE analysis of splicing of
MIHHsaWT. Splicing reactions consisted of 2.8 µM purified fusion protein in Buffer A
supplemented with 2 mM TCEP and 5 mM EDTA, with a final salt concentration of 2.0 M NaCl.
Splicing was initiated by incubation for 16 h at the temperatures indicated.
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Figure S5. Comparison of protein splicing of MIHHsa fusion proteins. Analysis of protein
splicing and N-terminal cleavage assayed by SDS-PAGE, stained by InVision His-tag in-gel
stain. (Compare to Coomassie Blue stained gel in Figure 2.) Splicing or cleavage was initiated by
incubation for 16 h at 28°C of 2.8 µM purified fusion protein in Buffer A with the final salt
concentration given and supplemented with 5 mM EDTA. Lanes labeled “P” are untreated
proteins. MIHHsaWT and MIHHsaQN were incubated with 2 mM TCEP, and MIHHsaQACA
with 150 mM DTT. Gel was stained with InVision His-tag in-gel stain; band identities in italics
(M and I) note where bands in Figure 2 are not visualized in Figure S5, as the proteins lack a