Site-specific Labeling of a Protein Lysine Residue By Novel Kinetic Labeling Combinatorial Libraries

Abstract

The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q) that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E) emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks.

Keywords: Affinity labels; Combinatorial libraries; Kinetic labeling libraries; Lysine labeling; Peptide libraries; Site-specific protein modification.

Figure 1

Reaction between a library member (1, J-OOXXE-NH₂) and the target protein (2, P-NH₂), involving transfer of the biotinyl function (B-C=O) with concomitant release of a 4-mercaptobenzoyl-pentapeptide (3).

Figure 2

Structure of the fastest labeling entity of HSA (7-E, FLYEE-NH₂) emerging from deconvolution of the pool of molecules of general structure, FLYOE-NH₂. Note that leucine at position 2 of the peptide possesses the D-configuration. The standard of comparison, (6), is shown lacking a peptidyl group.

Figure 3

Results of treating human serum albumin for one minute with each of 81 pools, of general structure J-OOXXE-NH₂, followed by quenching with 50 mM hydroxylamine. Sort values derived from ELISA ΔOD determinations (see experimental) are plotted on the z-axis for each pool, after subtracting the average sort value, 1.21. Thus Q (Y-axis) represents a pools’ sort value minus the average sort value of a library member. Residues at positions 1 and 2, are of the L- and D-configurations, respectively.

Figure 4

Results of a time course in which HSA was treated with each of nine pools of general structure J-FLOXE-NH₂ (5). The kinetic data are compared against a standard lacking a peptidyl affinity group (6), and the master pool of 81, denoted by J-FLXXE-NH2 (black plot). Only the J-FLYXE-NH₂ pool, (5-Y, red plot) substantially exceeded the rate of labeling of the master pool. (T_q refers to the time HSA was added to the quenched background reaction).

Figure 5

High resolution FTMS spectra of (A) native HSA, (B) HSA treated with one equivalent of J-FLYEE-NH₂ (7-E), (C), porcine serum albumin (PSA), (D) PSA treated with one equivalent of (7-E). These stoichiometric experiments of HSA and pig serum albumin (PSA) were carried out with HSA or PSA and (7-E) at 0.2 mM.

Figure 6

Evidence for biotin attachment at Lys-190, after HSA treatment with on equivalent of J-FLYEE-NH₂ (7-E). CID product ion spectrum from the precursor ion of m/z 872.931 (+2) present in LC-FTMS analysis of labeled HSA that has been digested with Lys C.

Figure 7

(A) High resolution FTMS spectra of native HSA, (B) HSA treated with one equivalent of J-FLYEE-NH₂ (9) after 1 hr, and (C) after 16 hr, (D) HSA treated with five equivalents of (9) after 1 hr, and (E) after 16 hr.

Figure 8

Schematic of minimal mechanisms of protein modification by a tight-binding affinity label, compared with a reactant that is not complementary to the protein target. The latter reaction is portrayed as a bimolecular reaction lacking both an intermediate and tight-binding interactions between the affinity group and protein.

Figure 9

A protein of m sites, treated with a library of n molecular shapes representing potential affinity labels, is shown schematically. Library members seek access to any complementary sites on the protein, which may result in transfer of Y to a proximal nucleophile.