Engineering the alpha(L) I domain.

Format:

Book

Thesis/Dissertation

Author/Creator:

Pepper, Lauren Rebecca.

Contributor:

University of Pennsylvania.

Language:

English

Subjects (All):

Biomedical engineering.

0541.

Local Subjects:

0541.

Physical Description:

119 pages

Contained In:

Dissertation Abstracts International 70-02B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

Lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta 2 integrin) mediates the adhesion of leukocytes by binding to intracellular adhesion molecule-1 (ICAM-1), which is upregulated on the endothelium at sites of inflammation. The inserted (I) domain of the alphaL subunit contains the entire ligand binding site of the molecule, and can exist in a number of conformations that can modulate behavior from rolling to firm adhesion. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High affinity mutants of the I domain have been discovered previously using both rational design and directed evolution.

Here we demonstrate the first example of an allosteric adhesive switch by engineering the alphaL I domain. The switch is regulated by ligand binding to secondary domains that flank the I domain and confer increased affinity of the I domain fusion for ICAM-1 upon peptide binding. Using directed evolution, the I domain fusion was refined to increase the switch activity. Yeast displaying this I domain fusion on the surface exhibit rolling adhesion along ICAM-1 surfaces under shear flow, and these yeast show a significant switch to firm adhesion upon addition of soluble target peptide.

We have also found that affinity is not the only parameter that dictates the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel sorting method to separate yeast expressing a library of I domain variants on the surface by affinity under flow. Using conditions analogous to those experienced by cells along the endothelium, we have identified mutations that support firm adhesion not found using typical directed evolution techniques that select for binding to soluble ligands. Firmly adherent mutants isolated using this method harbor mutations that do not exclusively cluster with those found sorting with soluble ligand for higher affinity. This analysis supports a greater understanding of the structure-function relationship of the alphaL I domain and the connection between force and adhesion.

Notes:

Source: Dissertation Abstracts International, Volume: 70-02, Section: B, page: 1167.

Advisers: Daniel A. Hammer; Eric T. Boder.

Thesis (Ph.D.)--University of Pennsylvania, 2008.

Local Notes:

School code: 0175.

ISBN:

9781109008593

Access Restriction:

Restricted for use by site license.