There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redoxinsensitive, cyclic peptides that target the signaling protein Gαi1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic Gαi binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity (K i ≈ 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the βγ heterodimer, an endogenous Gαi1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.Although networks of protein-protein interactions control function inside cells, it is increasingly clear that many of these players cannot be targeted using traditional druglike molecules (1). High-affinity, high-specificity ligands targeting protein surfaces are thus of considerable interest as tools for chemical genetics, as potential lead/surrogate compounds, and as new drugs. Nanotechnology could also greatly benefit from such molecules, particularly robust, inexpensive, low-molecular-weight ligands that could replace monoclonal antibodies (2). Designing these ligands still remains a significant challenge despite the tremendous advances in structural biology and computational chemistry.Our laboratory has been working to design new peptide ligands that target heterotrimeric Gprotein signaling. We have previously used messenger RNA (mRNA) display (3) to isolate new linear peptides that target Gαi1 (4-6). Gαi1 is a member of the Gα subunit family and serves as a molecular router, connecting the cell-surface G-protein-coupled receptors (GPCR) to down-stream effector pathways (see ref 7 for a review of GPCR signaling). Gα subunits function by collaboration with the Gβγ heterodimer and a transmembrane receptor. The Gαβγ heterotrimer associates with the cytosolic portion of GPCRs with GDP bound in the nucleotide pocket of Gα. Extracellular ligand binding to the GPCR results in
Peptides were generated on an Applied Biosystems 432A peptide synthesizer using solid phase, F-Moc chemistry. Crude peptides were deprotected by TFA/ethanedithiol/thioanisole treatment and purified on a C-18 reverse phase HPLC column to a final purity greater than 95% (MALDI-TOF, Analytical C-18 HPLC). Peptides without a naturally occurring tryptophan or tyrosine residue were synthesized with a carboxy-terminal Gly-Tyr tag for quantification purposes.Unlabeled RNA hairpins (λboxB R15 and P22boxB L15 ) were synthesized by in vitro transcription using T7 RNA polymerase. 1 The RNA was purified by 20% urea-PAGE, desalted on a NAP column (Amersham Pharmacia), and freeze-dried. RNA was quantified by UV absorption at a wavelength of 260 nm.Labeled RNA hairpins containing 2-amino purine (2AP) at loop position 2 (2AP-2), 3 (2AP-3), or 4 (2AP-4) were constructed by automated RNA synthesis using either 2-aminopurine-TOM-CE phosphoramidite or 2'-O-methyl 2-aminopurine phosphoramidite (Glen Research, Sterling, VA). Steady-State Fluorescence MeasurementsMeasurements were conducted following the procedures of Barrick et. al. 2 Titrations were performed on a ShimadzuSpectrofluorophotometer at 20º C with Excitation/Emission wavelengths at 310/370 nm . Peptides were titrated iteratively into a constantly stirred solution of 2AP labeled RNA hairpin (20-200 nM RNA). Binding buffer contained 20 mM Tris-OAc, with a variable concentration of KOAc (15 mM-500 mM) at pH 7.5. Binding Constants were calculated for a one step binding mechanism by nonlinear least squares regression using the computer program DynaFit. 3 All isotherms were fit with < 10% uncertainty. Stopped-Flow Fluorescence MeasurementsExperiments were conducted following the procedures of Lacourciere et al. 4 Measurements were performed at 20º C under standard buffer conditions (20 mM Tris-OAc, 50 mM KOAc, pH 7.5) using a stop-flow device from Applied Photophysics (Surray, U.K.) in twosyringe mode. Fluorescence excitation was performed at 310 nm and emmision was measured with a filter cutoff > 360 nm. The
N proteins from bacteriophages λ, P22, and φ21 modulate transcription elongation by binding nascent “boxB” mRNA hairpins. This RNA recognition is mediated by N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB RNA targets. Here, we have analyzed the affinity and specificity of the peptide−RNA interactions that modulate this transcriptional switch. To do this, we constructed a series of peptides based on the wild-type λ, P22, and φ21 N protein binding domains ranging from 11 to 22 residues and analyzed their interactions with the leftward and rightward boxB RNA hairpin targets for all three phage. Binding constant (K d) values were determined using RNA hairpins labeled with 2-aminopurine (2AP) and monitoring the fluorescence change as peptide was added. K d's demonstrate that λ and P22 N peptides bind to their cognate boxB targets with high specificity and show equal affinities for their leftward and rightward hairpins. Surprisingly, φ21 shows very little specificity for its cognate targets. λ and P22 N peptides exhibit differential modes of recognition with specificity conferred by their amino- and carboxy-terminal modules, respectively. We have generated a reciprocal matrix of substituted peptides to examine the contributions of individual residues to specificity. Amino acid coupling analysis supports a binding model where the Arg8 residue of λ peptide acts as a conformational hot spot, anchoring the induced loop fold of its boxB hairpin target.
Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G␣ i1 . The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of G␣ i1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here, we demonstrate that the R6A-1 peptide interacts with G␣ subunits representing all four G protein classes, acting as a core motif for G␣ interaction. This contrasts with the consensus G protein regulatory (GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (G␣ i/o -Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to G␣ i1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G␣ subunits and excludes association with G␥. R6A-G␣ i1 complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg 2؉ , suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using G␣ i1 /G␣ s chimeras identify two regions of G␣ i1 (residues 1-35 and 57-88) as determinants for strong R6A-G i␣1 interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.Heterotrimeric guanine nucleotide-binding proteins (G proteins), composed of ␣, , and ␥ subunits, mediate signaling from transmembrane receptors (GPCRs) 3 to a wide variety of effectors (1, 2). In the inactive state, intracellular G␣-GDP is tightly bound to G␥ to form a G␣␥ heterotrimer. Activation of a GPCR results in GDP exchange with GTP in the G␣ subunit, concomitant dissociation of G␥, and subsequent signal transduction through G␣-GTP and/or G␥. The inherent guanosine triphosphatase (GTPase) activity of G␣, which is accelerated by various GTPase-activating proteins, returns the protein to the GDP-bound state, resulting in reassociation with G␥ and termination of signaling.Approximately 50% of currently marketed drugs target cell surface-accessible GPCRs (3, 4). Drug discovery targeting intracellular G proteins directly is inherently difficult due to (i) the broad spectrum of signaling events mediated at the G protein level, (ii) the requirement that drugs must cross the cell membrane to reach intracellular G proteins, and (iii) the high sequence and structural similarities between G protein classes (5, 6). Nevertheless, a number of diseases have been attributed to aberrant G protein activity (7,8), and direct G protein ligands will provide new approaches and selectivities for drug treatment (5, 6, 9).Selection methodologies facilitate the isolation of rare molecules with unique functions from large libraries (10, 11). Selections with combinatorial libraries have already been used to identify peptides that bind to various proteins in the G protein signaling cycle (9). We re...
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