Determination of Hemin‐Binding Characteristics of Proteins by a Combinatorial Peptide Library Approach

Abstract

Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron-ion-containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate-covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed. Ligands for both types of binding have been reported independently, though sometimes with different affinities for similar sequences. We applied a combinatorial approach using the library (X)(4) (C/H/Y)(X)(4) to characterize peptide ligands with considerable hemin binding capacities. Some of the library-selected peptides were comparable in terms of hemin association independently of whether or not a cysteine residue was present in the sequence. Indeed, a preference for His-based (≈39 %) and Tyr-based (≈40 %) sequences over Cys-based ones (≈21 %) was detected. The binding affinities for the library-selected peptides, as determined by UV/Vis spectroscopy, were in the nanomolar range. Moreover, selected representatives efficiently competed for hemin binding with the human BK channel hSlo1, which is known to be regulated by heme through binding to its heme-binding domain.

A heme-binding domain controls regulation of ATP-dependent potassium channels

“…In the case of ion channel regulation, it has recently emerged that there is a role for heme (12,(23)(24)(25)(26), but the observations are largely empirical, so that the molecular basis for the regulatory control within individual channel proteins has yet to be properly defined. A number of heme-responsive motifs (27) have been suggested to be involved: these include Cys/Pro (CP) motifs using thiolate ligation to the heme as in the P450s or Cys/His motifs (3,14). In the case of the Slo1 channels (12), a cytochrome c-like CXXCH motif has been implicated (12,28).…”

Prevalence and Gene Characteristics of Antibodies with Cofactor-induced HIV-1 Specificity

“…ing promiscuity of heme. Indeed, computational and experimental studies have revealed that heme-binding motifs on distinct proteins are characterized by a vast diversity of sequences and structures (44,45). We hypothesized that heme could interact with Abs in many alternative ways, depending on the sequence of the variable region.…”

Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM)

“…As a possible way to investigate this question, different peptide libraries were synthesized and screened for heme binding properties [4,10]. In particular, a combinatorial peptide library revealed useful information about the influence of the coordinating amino acid and the flanking regions on the heme-binding properties [4].…”

“…Well-studied examples are the heme-dependent regulation of gating in the human big potassium (BK) channel Slo1 [2][3][4][5] and the iron response regulator (Irr) [6,7]. Another example is the nuclear hormon receptor Rev-erbβ, where the Fe(III)-heme is either five-or sixfold coordinated depending on the redox conditions [8].…”

“…The results of these efforts were compiled into lists of short sequence motifs, so-called potential "heme regulatory motifs" (HRMs). The HRMs were further grouped based on features in their amino acid sequence [4,11,12]. The so far best-studied HRM class contains the "CP motif", i.e.…”