The role of buried stack residues in the folding of the beta-helix domain of P22 tailspike

In-register, parallel alignment of similar or identical side chains is a common structural phenomenon in amyloid fibers. In the crystal structure of an amyloid fiber, these residues not only align, but take identical side chain orientations, creating long stacks of identical residues with identical orientations. This phenomenon of side chain stacking is a dominant component of the amyloid structure, and may take an equally important role in the seemingly sequence-independent formation of amyloid fibers or the sequence-dependent species barrier to amyloid transmission. Side chain stacking has long been observed as a prominent feature of the soluble parallel [beta]-helix fold, which exhibits a cross-[beta] structure reminiscent of amyloids. To investigate the sequence requirements of these stacks for directing a polypeptide chain into a [beta]-helical fold, we performed systematic mutational studies using the complete P22 tailspike protein, which contains a 13 rung [beta]-helix domain. The in vivo folding and assembly of 150 single mutant polypeptide chains were characterized at multiple temperatures by SDS-PAGE, which distinguishes the fully native, SDS-resistant state from partially folded or misfolded conformers. The vast majority of the buried core was completely intolerant to substitution at physiological temperatures, while only a few sites composing a continuously contacting network of residues called the folding spine were intolerant of alanine mutations at 18⁰C. These results indicate that the [beta]-helix folds through a processive folding mechanism, analogous to the nucleation and elongation mechanism of amyloids, but requiring recurring sequence-dependent stacking contacts that stretch the length of the [beta]-helix. Numerous avenues of investigation demonstrated that the observed folding defects of the full length tailspike were due to a sequence effect on the folding of the [beta]-helix domain.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Simkovsky, Ryan Matthew

Advisor dc:contributor.advisor

• Jonathan King.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/40866

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/40866