Probing the Ligand‐Binding Specificity and Analyzing the Folding State of SPOT‐Synthesized FBP28 WW Domain Variants

Abstract

The WW domains are known as the smallest naturally occurring, monomeric, triple-stranded, antiparallel beta-sheet domains. Hence, we chose the FBP28 WW domain as a model to investigate the stability of the beta-sheet structure at the amino acid level in the context of its function (ligand binding). The structure-function relationship was investigated through a complete substitution analysis of the FBP28 WW domain, with variants synthesized as a cellulose-bound peptide array. The functionality of the FBP28 WW domain variants was examined by probing the peptide array for ligand binding. In addition, selected FBP28 WW domain variants were investigated by CD measurements to determine the stability of the antiparallel beta-sheet structure. We discuss the correlation between structure stability and functionality for the FBP28 WW domain, as well as the effect of ligand-induced structure stabilization.

Synthetic peptide arrays for investigating protein interaction domains

“…This is in good agreement with Molina and co-workers reporting peptide purity in the range of 74.4-91.3% [63]. Even longer peptides such as the 34-meric FBP28 WW domain could be SPOT-synthesized with a high quality of 65% purity [64]. Besides the high synthetic peptide quality, equivalent peptide array quality is achieved by applying identical chemical conditions during array synthesis.…”

Mapping Putative Contact Sites Between Subunits in a Bacterial ATP-binding Cassette (ABC) Transporter by Synthetic Peptide Libraries

“…20,23 More recently, it was demonstrated that even the 34meric FBP28D15N WW domain could be SPOT synthesized with an overall purity of 65%. 24 The highly parallel character of the SPOT synthesis approach, the good synthesis quality of so prepared soluble peptides and the preparation of huge numbers of peptides at low costs make them ideal candidates for biological or biochemical solution-based screening studies. SPOT-synthesized soluble peptides have been successfully applied to several biological assay systems like T-cell epitope mapping, 22 or membrane-integral receptor studies like the PACAP-VIP interaction.…”

Partially Folded Bovine Pancreatic Trypsin Inhibitor Analogues Attain Fully Native Structures when Co-Crystallized with S195A Rat Trypsin

“…[6][7][8][9][10][11][12][13][14][15][22][23][24][61][62][63] While these proteins are disordered and very flexible in the 'free' state, they function biologically by adopting various folded structures upon binding to different kinds of cognate biological partners, such as small molecule effectors, substrates, cofactors, other proteins, nucleic acids, membranes, etc. 10,22,[64][65][66][67][68][69][70][71][72][73][74][75][76] The capability to undergo function-related disorder-order transitions may be advantageous, since it provides greater flexibility while retaining high specificity in the interaction/recognition with different biological targets, and it facilitates clearance by proteasemediated degradation once the functional purpose is served.…”