How to build disulfide bond between two cysteine, i.e. change two “-SH” (sulfydryl) into “-S-S-“?

[Anonymous]

Dear friends,
I have a Fab (antibody fragment of antigen binding) with two chains(LC 1-214, HC 215-442). An interchain disulfide bond is supposed to form between LC-214-cysteine and HC-434-cysteine. I am using 4KMT as its homology template. In the 4KMT structure, the interchain disulfide bond does exist. However, after replacing the residues with my own residues, the interchain disulfide bond has lost and become “-SH” instead of “-S-S-“.

(During the residue replacment, fixbb was used to replace residues in LC (light chain); remodel was to fill the missing residues in HC (heave chain) and minirosetta was used to replace residues in HC.)

So what is the best way to build disulfide bond between cysteine?

---

It seems that remodel can do this. What confuses me is, in the section “Disulfide design” on https://www.rosettacommons.org/docs/latest/rosettaremodel.html, none of the example is to connect two cysteine. I think only cysteine can form disulfide bond. But I still tried and the following is my unsuccessful results:

~/Cheng/rosetta_2014.30.57114_bundle/main/source/bin/remodel.linuxgccrelease -database ~/Cheng/rosetta_2014.30.57114_bundle/main/database -s /home/lanselibai/Cheng/20150117_disulfide/C226S.pdb -remodel:blueprint /home/lanselibai/Cheng/20150117_disulfide/blueprint_disulfide_build -remodel:quick_and_dirty -remodel:num_trajectory 1 -overwrite -remodel:use_pose_relax true -remodel:build_disulf true &> remodel.log &

# Trial 1
)blueprint:
…
214 C L DM_start DM_stop
215 E L
216 V L
…
434 C . DS_start DS_stop
…
)result:
residue C, E, V all become V, V, V (But I want to keep the residue types fixed)

# Trial 2
)blueprint:
…
214 C L DM_start DM_stop
215 E L PIKAA E
216 V L PIKAA V
…
434 C . DS_start DS_stop
…
)result:
residue C, E, V all become V, V, V (But I want to keep the residue types fixed)

# Trial 3
)blueprint:
…
214 C L DM_start DM_stop
…
434 C . DS_start DS_stop
…
)result:
“-SH” (sulfydryl) still exist in the cysteine residue, i.e. the exactly same as the input.

Thank you very much.

Yours sincerely
Cheng

[Anonymous]

I also read the “RosettaAntibody3” protocol. However, it is only for Fv region. As we know, the Fab contains Fv region and constant region (i.e. CH1 and CL region). So the interchain disulfide bond issue has not been considered. Could someone help me to deal with the interchain disulfide bond between light chain and heavy chain in Fab? Thank you very much.

[Author]

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