Harnessing the Unique Structural Properties of Isolated α-Helices

Abstract

The ␣-helix is a ubiquitous secondary structural element that is almost exclusively observed in proteins when stabilized by tertiary or quaternary interactions. However, beginning with the unexpected observations of ␣-helix formation in the isolated C-peptide in ribonuclease A, there is growing evidence that a significant percentage (0.2%) of all proteins contain isolated stable single ␣-helical domains (SAH). These SAH domains provide unique structural features essential for normal protein function. A subset of SAH domains contain a characteristic ER/K motif, composed of a repeating sequence of ϳ4 consecutive glutamic acids followed by ϳ4 consecutive basic arginine or lysine (R/K) residues. The ER/K ␣-helix, also termed the ER/K linker, has been extensively characterized in the context of the myosin family of molecular motors and is emerging as a versatile structural element for protein and cellular engineering applications. Here, we review the structure and function of SAH domains, as well as the tools to identify them in natural proteins. We conclude with a discussion of recent studies that have successfully used the modular ER/K linker for engineering chimeric myosin proteins with altered mechanical properties, as well as synthetic polypeptides that can be used to monitor and systematically modulate protein interactions within cells. Coiled-coil or Single ␣-Helical (SAH) Domain?Until recently, SAH 2 domains in natural proteins were predicted by secondary structure prediction algorithms to form a coiled-coil, in part due to the high concentration of charged and polar residues that are also the hallmark of the coiled-coil motif (1). Among the multiple folds in globular proteins that stabilize ␣-helices, the coiled-coil motif has been extensively characterized and in general is the most predictable form of tertiary protein structure (2). In the coiled-coil motif, two or more ␣-helices are individually stabilized by sequence-specific packing at consensus hydrophobic patches. Extensive studies have elicited general sequence and structure rules that govern coiled-coil interactions. Briefly, the amino acid sequence of each ␣-helix in a coiled-coil is divided into heptads (7 residues) that form nearly two complete ␣-helical turns and span 1.05 nm along the helical axis. Each amino acid in the heptad is described by its relative position, moving from the N to C terminus, using the nomenclature abcdefg. In canonical dimeric coiled-coils, the a and d positions radiate away from the core of the ␣-helix, 60°a part and offset by 0.45 nm along the helix length, and are typically occupied by aliphatic hydrophobic residues, whereas polar residues comprise the rest of the positions. As the heptad is repeated, it forms a continuous hydrophobic patch located along a single face of the ␣-helix compatible with a tight intermolecular interaction between polypeptides with the same or similar heptad pattern (2). However, often polar or charged residues occupy the a and d positions, leading to local destabilization of the coiled-coil...

Single-chain lanthanide luminescence biosensors for cell-based imaging and screening of protein-protein interactions

“…Because the affinity elements are tethered together, their effective concentration depends only on linker length and is independent of7 solution concentration. The overall fraction of an ER/K biosensor in the closed or On-state depends only on the K D of the affinity elements and linker length.Consequently, protein-protein interactions may be observed and analyzed even when the overall sensor concentration is far below the K D31 .TGL microscopy of biosensors in live cells. NIH3T3 fibroblasts were stably transfected with plasmid DNA encoding a single fusion protein under control of a Tet-responsive promoter that contained the following elements (from N-to C-terminus): FRB, eDHFR, ER/K, GFP and FKBP12 (Figure 1).…”

Functional and Structural Characterization of Novel Type of Linker Connecting Capsid and Nucleocapsid Protein Domains in Murine Leukemia Virus

“…SAHs are rich in arginine, lysine, and glutamate residues. SAHs are isolated ␣-helices that are stable without being a part of a globular protein or a coiled-coil, and whose stability is thought to be achieved by intrahelical electrostatic interactions between arginine and glutamate residues, or lysine and glutamate residues (24,25). SAHs are usually 30 -200 residues long, and often serve as linkers bridging two protein domains.…”

Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor

“…This "catalytic core" of the C1a and C2a domains of ACs has been demonstrated to have a weak affinity between the two domains (∼10 mM) and is sufficient to reconstitute cAMP generation (Tang and Gilman, 1995). In the SYNAC sensor, these domains are separated by a modular 30 nm ER/K linker that provides weak basal complementation (effective concentration ∼100 nM) between the domains (Swanson and Sivaramakrishnan, 2014) as well as providing a substantial separation between the two sets of fluorophores used for the FRET readout (Fig. 1A).…”

“…Interactions between the domains flanking the ER/K linker bring the FRET pair into close proximity, leading to a measurable increase in FRET (Swanson and Sivaramakrishnan, 2014). In addition, effector-dependent complementation of the C1 and C2 domains stimulates cAMP generation, which can be measured using established assays.…”