Understanding Metalloprotein Folding Using a de Novo Design Strategy

Abstract

Metal ions play significant roles in most biological systems. Over the past two decades, there has been significant interest in the redesign of existing metal binding sites in proteins/peptides and the introduction of metals into folded proteins/peptides. Recent research has focused on the effects of metal binding on the overall secondary and tertiary conformations of unstructured peptides/proteins. In this context, de novo design of metallopeptides has become a valuable approach for studying the consequence of metal binding. It has been seen that metal ions not only direct folding of partially folded peptides but have at times also been the elixir for properly folding random-coil-like structures in stable secondary conformations. Work in our group has focused on binding of heavy metal ions such as Hg(II) to de novo designed alpha-helical three stranded coiled coil peptides with sequences based on the heptad repeat motif. Removal from or addition of a heptad to the parent 30-residue TRI peptide with the amino acid sequence Ac-G(LKALEEK)(4)G-NH(2) generated peptides whose self-aggregation affinities were seen to be dependent on their lengths. It was noted that adjustment in the position of the thiol from an "a" position in the case of the shorter BabyL9C to a "d" position for BabyL12C resulted in a peptide with low association affinities for itself, weaker binding with Hg(II), and a considerably faster kinetic profile for metal insertion. Similar differences in thermodynamic and kinetic parameters were also noted for the longer TRI peptides. At the same time, metal insertion into the prefolded and longer TRI and Grand peptides has clearly demonstrated that the metal binding is both thermodynamically as well kinetically different from that to unassociated peptides.

Protein Design: Toward Functional Metalloenzymes

“…The heptad is composed of seven amino acid residues, denoted as abcdefg , where a and d positions are usually hydrophobic residues, and e and g positions are salt-bridging residues. 704,705 The connectivities between helices and overlapping hydrophobes are often represented as helical wheel diagrams (Figure 29). For heptads containing leucine, the addition of each 7 amino acid repeat confers ~9 kcal/mol of stability to the system.…”

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

“…[19] These studies demonstrated that modification of steric bulk around the metal-binding site could control solvent/substrate access to the metal. The first generation CuNiR model peptide, TRI W-H, contains a Leu residue at the d -sites [20] above and below the Cu-binding histidines in the 23 rd position, in a fashion analogous to TRIL16C. Therefore, we substituted the Leu residues at the 19 th and 26 th positions in three different ways.…”

Using Nonnatural Amino Acids to Control Metal‐Coordination Number in Three‐Stranded Coiled Coils

“…[4] In particular, we have asked whether the same metal can be bound to structurally related peptides with identical first-coordinationsphere ligands derived from the protein, but in one case form a trigonal structure and in a second case form a fourcoordinate pseudo-tetrahedral environment. [4] The relative importance of a metals geometric preference and the inherent protein structure is fundamental to understanding the folding, [5][6][7][8] stability, [9,10] and conformational changes [4,5,11] of metalloproteins, and hence the control of such site discrimination has been an important objective of protein design studies. The peptides described herein are variants of the TRI family of peptides shown in Table 1.…”