The Most Stable Protein–Ligand Complex: Applications for One‐Step Affinity Purification and Identification of Protein Assemblies

Inhibiting Aggregation of β-Amyloid by Folded and Unfolded Forms of Fimbrial Protein of Gram-Negative Bacteria

“…The mode of interaction between this donated strand and its acceptor is termed ‘donor strand complementation’ (DSC) . This interaction forms a highly stable noncovalent bond interaction that has previously been used as a handle for protein purification . Structural analyses,, show that DSC is achieved through hydrogen‐bond interactions to form a β‐sheet as well as through hydrophobic interactions .…”

“…In initial attempts, a gene (see the Supporting information, SI) was cloned that encodes the strand acceptor part of FimG of Escherichia coli K12 strain (Gene Accession Number: AP009048), termed FimGt (Figure ; ‘t’ denotes ‘truncated’ to show that it lacks the N‐terminal donor strand of its own). When the protein was overexpressed by IPTG induction, FimGt was only found in the insoluble cell debris after lysis of the bacteria.…”

“…The insoluble proteins were dissolved in 6 M guanidinium hydrochloride (GdmHCl) and FimGt was purified by immobilized metal affinity chromatography (IMAC) in denatured form. The protein was then refolded by using a rapid dilution procedure . The refolded FimGt was subjected to gel filtration and three peaks were observed in the chromatogram (Figure a), the first and the last of which were collected; the corresponding proteins were termed ‘Poly’ and ‘Mono’, respectively, based on the molecular weight estimation from the elution time.…”

Accelerating the Association of the Most Stable Protein–Ligand Complex by More than Two Orders of Magnitude

“…[6b] Theb imolecular complex between FimGt, an N-terminally truncated FimG variant lacking its own donor strand, and the DsF peptide has adissociation constant (K D )o f1 .5 10 À20 m.T he interaction is dominated by an extremely low dissociation rate (k off )of510 À18 s À1 which can only be observed when FimGt unfolds in conjunction with peptide release. [6] Nevertheless,the association of FimGt with DsF proved to be slow (k on = 330 m À1 s À1 )c ompared to most natural protein-protein or protein-peptide complexes,which is likely ac onsequence of the fact that pilus subunit association is catalyzed by the assembly platform FimD in vivo. [8] Our approach for improving the k on value of the FimGt/DsF complex through amplified electrostatic attraction was based on the crystal structure of the complex (Figure 1).…”

“…TheF imG/DsF complex is the thermodynamically and kinetically most stable,n oncovalent protein-ligand complex reported to date. [6] Thei nteraction between FimG and DsF follows the mechanism of donor strand complementation, in which the incomplete,i mmunoglobulin-like fold of every pilus subunit is completed by an Nterminal extension, termed donor strand (Ds), of the following subunit. [7] This type of interaction leads to virtually infinite stability of the pilus against spontaneous dissociation and unfolding.…”