Hinge-Motion
Binding Proteins: Unraveling Their Analytical Potential Hinge-motion binding proteins undergo a conformational change that can be used as the basis for quantifying analytes.
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hemical analysis has benefited significantly from recent advances in instrumentation that enhance detection sensitivity. The complexity of sample matrices and the presence of compounds structurally related to an analyte dictate the need for sensitive and selective methods that can discriminate the analyte from interferences. Research has focused on the discovery of synthetic molecular recognition systems, referred to as host–guest chemistry, as well as the identification of biological recognition systems. Among such biomolecules, hinge-motion binding proteins (HMBPs) demonstrate exquisite selectivity toward their corresponding ligand/analyte, with affinities KD down to the nanomolar range. These proteins are so named because they undergo a significant conformational change upon binding of the ligand—the two domains bend around a “hinge” region of the protein. The binding mechanism of these proteins resembles that of the carnivorous plant commonly known as the Venus flytrap. The conformational change that HMBPs undergo upon binding of a ligand can be used to quantify a target analyte. In one approach, a reporter group, such as a fluorescent probe, is strategically positioned on the protein so that it experiences a change in its microenvironment as a result of a binding event (Figure 1a). Another method involves protein chimeras, in which the HMBP is fused with other proteins and the signal is transduced upon binding with the target analyte (Figure 1b). In a third technique, the HMBP can be labeled with a reporter molecule, which moves as a result of the binding event either closer to or further away from the signal transducer (Figure 1c). A fourth strategy uses the Elizabeth A. Moschou conformational change to enable the expression of a reporter molecule Leonidas G. Bachas through a transcriptional regulation mechanism (Figure 1d). Sylvia Daunert Different classes of HMBPs include periplasmic binding proteins; transcriptional regulators; enzymes; and messenger proteins, like calUniversity of Kentucky modulin (1). This article focuses on periplasmic binding proteins and Sapna K. Deo calmodulin, because they have already been applied to environmental Indiana University–Purdue and metabolite monitoring, food technology, bioanalysis, high-throughUniversity Indianapolis put screening, and diagnostics. © 2006 AMERICAN CHEMICAL SOCIETY
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each other by two or three pliable peptide strands that form the hinge. The two hinged doFRET mains form a cleft, which constitutes the ligand binding site (c) (d) d of the protein (5). When the NA stran D O2 O2 protein is “open”, the two doe– – e mains of the protein lie apart, exposing the binding site and I1 I1 making it accessible to the Arabinose I 2 I2 Analyte entry or exit of the ligand. pBAD pBAD When the protein binds to the No transcription Transcription ligand, the two domains come closer to one another at the FIGURE 1. Design strategies of sensing systems based on HMBPs. hinge; the angle between them (a) Interaction of the protein with the analyte alters the fluorescence signal of a response probe, which is can change by as much as 60o strategically positioned on the protein. (b) Interaction of the analyte with the HMBP enables FRET between (6) (Figure 2). This conformatwo fluorescent probes, located at the two termini. (c) Strategic positioning of a reporter molecule on the tional change occurs mostly at HMBP, which upon binding moves away from the signal transducer. (d) Binding event of the target analyte to the HMBP enables the transcription and expression of a reporter molecule. One example is the arabithe hinge, with no significant nose induction system, in which I1, I2, and O2 are binding sites and pBAD is the promoter DNA region. alteration to the conformation of the two domains per se. During the binding event, a series of interactions occurs between the ligand and the amino acids that are part of the bindA closer look The sulfate binding protein from Salmonella typhimurium was ing site of the protein. For example, the cleft of the E. coli nickthe first periplasmic binding protein to be identified, in 1965. el binding protein (NBP) is nonpolar and neatly fits the pentaThese proteins, found in the periplasm of Gram-negative bacte- coordinate-hydrated nickel cation (7). The selectivity of NBP to ria, serve as high-affinity binders/receptors for the uptake and Ni2+ is based on cation– interactions as well as the coordination transport of specific nutrients from the periplasm to the cyto- geometry of nickel. In the case of E. coli phosphate binding proplasm of cells (2). In general, they are relatively small proteins, tein (PBP), the binding pocket itself is negatively charged, but with masses in the range 25–59 kDa. So far, >100 periplasmic positively charged patches on the top and bottom of the entrance binding proteins have been isolated and characterized from a va- of the binding cleft attract the phosphate anion. After the phosriety of sources, including E. coli, thermophilic bacteria, and eu- phate anion enters the binding pocket, it is then stabilized by 12 karyotes (3). These proteins are generally categorized by the type hydrogen bonds and by electrostatic attraction with the positiveof corresponding ligands, that is, into the classes of periplasmic ly charged Arg135 (8). binding proteins that are selective for metals, oxoanions, carboFor the saccharide binding proteins, such as glucose binding hydrates, amino acids, vitamins, and oligopeptides (Table 1). protein (GBP) and maltose binding protein (MBP), the sacchaAlthough the sequence homology between the various ride ligands are stabilized in the protein binding pocket by a seperiplasmic binding proteins is quite low, their 3D structures are ries of interactions, including hydrogen bonds, van der Waals inprominently similar. All these proteins are composed of two teractions, and salt bridges. The high affinity and selectivity of globular domains, one representing the amino-terminal domain these proteins for their respective ligands is based on the direc(N-domain) and the other the carboxy-terminal domain (C-do- tional nature of these bonds, steric hindrance, and the favorable main). Depending on their structure, periplasmic binding pro- polar environment of the binding pocket. In the amino acid teins can be categorized into two structural classes. Class I pro- binding proteins, the amino acids are stabilized inside the cleft teins are composed of 6 strands, and those in class II have a mostly through hydrogen bonding, although salt bridges and structure containing 5 strands (4). Periplasmic binding pro- hydrophobic interactions further assist the binding of the ligand. teins are thought to be derived from a common ancestor because The specificity of these proteins for their amino acid ligands is of the similarity in their 3D structure, ligand binding mecha- due to the directional nature of the hydrogen bonds and is also nism, and operon organization; however, their central -sheet tailored by the small conformational changes that selected rescore structure is different. This variation is due to a process called idues within the binding pocket undergo in order to achieve a “domain dislocation”, which involves intramolecular exchange geometric fit and favorable interaction with the ligand. In the of strands between the two domains of the periplasmic binding case of the nonperiplasmic binding protein calmodulin, pheproteins. The exchange results in the spatial rearrangement of nothiazines and tricyclic antidepressants cause a hinging motion secondary structural elements and is responsible for the observed in the presence of Ca2+ by binding mostly through van der Waals differences in the crystal structures (2, 4). interactions with a hydrophobic pocket of the protein (Figure 2 Typically, the two domains of these proteins are tethered to bottom). 6694
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Table 1. Applications of HMBPs. Putting HMBPs to work
Class
HMBPs
KD (µM)
Ions
Sulfate
0.1
Applications Sulfate sensing (14 )
The inherent ability of HMBPs to Phosphate 0.1 Phosphate detection undergo reversible conformational (16, 17, 32–34) changes upon binding allows for the Nickel
