Miniature Protein Research Articles

β‐Peptides as Inhibitors of Protein—Protein Interactions

AbstractFor Abstract see ChemInform Abstract in Full Text.

Paralog-Selective Ligands for Bcl-2 Proteins

There is considerable current interest in molecules that bind intra- or extracellular protein surfaces and inhibit protein-protein interactions. Previously we have reported that miniature proteins based on pancreatic-fold polypeptides can recognize even shallow alpha-helix binding clefts with high affinity and selectivity against unrelated proteins. One such miniature protein, PPBH3-1, binds the anti-apoptotic protein paralogs Bcl-2 and Bcl-XL with nanomolar affinity and a DeltaDeltaG = 1.2 kcal.mol-1 preference for Bcl-XL. Here we describe the directed evolution of PPBH3-1 into two new miniature proteins, PPBH3-5 and PPBH3-6, whose paralog specificity is reversed relative to PPBH3-1. PPBH3-5 and PPBH3-6 bind Bcl-2 with nanomolar affinity and a DeltaDeltaG = 0.9-1.3 kcal.mol-1 preference for Bcl-2 over Bcl-XL. Experiments with Bcl-XL variants suggest that PPBH3-5 and PPBH3-6 achieve high paralog specificity by exploiting subtle structural or electrostatic differences in the Bcl-2 and Bcl-XL molecular landscapes. PPBH3-5 and PPBH3-6 may have unique applications as early examples of nonnatural ligands that interact selectively with Bcl-2 proteins.

β-Peptides as inhibitors of protein–protein interactions

We became interested several years ago in exploring whether 14-helical β-peptide foldamers could bind protein surfaces and inhibit protein–protein interactions, and if so, whether their affinities and specificities would compare favorably with those of natural or miniature proteins. This exploration was complicated initially by the absence of a suitable β-peptide scaffold, one that possessed a well-defined 14-helical structure in water and tolerated the diverse sequence variation required to generate high-affinity protein surface ligands. In this perspective, we describe our approach to the design of adaptable β-peptide scaffolds with high levels of 14-helix structure in water, track the subsequent development of 14-helical β-peptide protein–protein interaction inhibitors, and examine the potential of this strategy for targeting other therapeutically important proteins.

A stable miniature protein with oxaloacetate decarboxylase activity.

An 18-residue miniature enzyme, Apoxaldie-1, has been designed, based on the known structure of the neurotoxic peptide apamin. Three lysine residues were introduced on the solvent-exposed face of the apamin alpha-helix to serve as an active site for decarboxylation of oxaloacetate. The oxidised form of Apoxaldie-1, in which two disulfide bonds stabilise the alpha-helix, formed spontaneously. CD spectroscopy measurements revealed that, in its oxidised form, Apoxaldie-1 adopted a stably folded structure, which was lost upon reduction of the disulfide bonds. Despite its small size and the absence of a designed binding pocket, Apoxaldie-1 displayed saturation kinetics in its oxidised form and catalysed the decarboxylation of oxaloacetate at a rate that was almost four orders of magnitude faster than that observed with n-butylamine. This rivals the performance of the best synthetic oxaloacetate decarboxylases reported to date. Unlike those, however, Apoxaldie-1 displayed significant stability. It maintained its secondary structure at temperatures in excess of 75 degrees C, in the presence of high concentrations of guanidinium chloride and at pH values as low as 2.2. Apamin-based catalysts have potential for the generation of miniature peptides that display activity under nonphysiological conditions.

From Transcription Factors to Designed Sequence‐Specific DNA‐Binding Peptides

AbstractFor Abstract see ChemInform Abstract in Full Text.

High affinity, paralog-specific recognition of the Mena EVH1 domain by a miniature protein.

Many protein domains involved in cell signaling contain or interact with proline-rich sequences, and the design of molecules that perturb signaling pathways represents a foremost goal of chemical biology. Previously we described a protein design strategy in which the well-folded alpha-helix in avian pancreatic polypeptide (aPP) presents short alpha-helical recognition epitopes. The miniature proteins designed in this way recognize even shallow protein clefts with high affinity and specificity. Here we show that the well-folded type-II polyproline helix in aPP can present the short PPII-helical recognition epitope within the ActA protein of Listeria monocytogenes. Like miniature proteins that use an alpha-helix for protein recognition, the miniature protein designed in this way displays high affinity for a natural ActA target, the EVH1 domain Mena1-112, and achieves the elusive goal of paralog specificity, discriminating well between EVH1 domains Mena1-112, VASP1-115, and Evl1-112. Most importantly, the miniature protein competed with ActA in Xenopus laevis egg cytoplasmic extracts, decreasing actin-dependent motility of L. monocytogenes and causing extreme speed variations and discontinuous tail formation. Our results suggest that miniature proteins based on aPP may represent an excellent framework for the design of ligands that differentiate the roles of EVH1 domains in vitro and in vivo.

Molecular recognition of protein surfaces: high affinity ligands for the CBP KIX domain.

Potent and specific inhibitors of protein.protein interactions have potential both as therapeutic compounds and biological tools, yet discovery of such molecules remains a challenge. Our laboratory has recently described a strategy, called protein grafting, for the identification of miniature proteins that bind protein surfaces with high affinity and specificity and inhibit the formation of protein.protein complexes. In protein grafting, those residues that comprise a functional alpha-helical binding epitope are stabilized on the solvent-exposed alpha-helical face of the small yet stable protein avian pancreatic polypeptide (aPP). Here we use protein grafting in combination with molecular evolution by phage display to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. Furthermore, we show that grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold yields molecules capable of high affinity recognition of CBP KIX even in the absence of phosphorylation. Importantly, both classes of designed ligands exhibit high specificity for the target CBP KIX domain over carbonic anhydrase and calmodulin, two unrelated proteins that bind hydrophobic or alpha-helical molecules that might be encountered in vivo.

Miniature homeodomains: high specificity without an N-terminal arm.

Recently, we described a strategy for the design of miniature proteins that bind DNA and protein surfaces with high affinity and selectivity. This strategy involves identifying the functional epitope required for macromolecular recognition by a natural protein and presenting it on a small, stable protein scaffold. In previous work, high-affinity DNA recognition was achieved only when the miniature protein contained the complete functional epitope. Here we report a miniature homeodomain that recognizes its 6-bp target site in the nanomolar concentration range at 25 degrees C, despite the absence of DNA contact residues located along the homeodomain N-terminal arm. We conclude that miniature proteins can achieve high affinity and selectivity for DNA by design even when the functional epitope is incomplete by using pre-organization to effectively compensate for lost protein-DNA contacts. In this case it has been possible to miniaturize both the recognition surface and the structural framework of a globular protein fold.

Rapid Cooperative Two-state Folding of a Miniature α–β Protein and Design of a Thermostable Variant

There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed α–β protein (NTL9 1–39) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20 mM sodium acetate, 100 mM NaCl the temperature of maximum stability is 6 °C, the t m is 65.3 °C, Δ H°( t m) is between 24.6 kcal mol −1 and 26.3 kcal mol −1, and Δ C p° is 0.38 kcal mol −1 deg −1. The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/ 2H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640 s −1 and the value of Δ G° calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t m of 82 °C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state.

From transcription factors to designed sequence-specific DNA-binding peptides.

Transcription factors are DNA-binding proteins responsible for initiating the transcription of particular genes upon interacting with specific DNA sequences located at their promoter or enhancer regions. The DNA recognition process, which is extremely selective, is mediated by non-covalent interactions between appropriately arranged structural motifs of the protein and exposed surfaces of the DNA bases and backbone. The great variability in DNA recognition by transcription factors has hampered the characterization of an amino acid-base step recognition code, making it very difficult to design non-natural peptides that can mimic the DNA-binding properties of these naturally occurring counterparts. However, in recent years, several transcription factor-based miniature proteins capable of tight interaction with specific DNA sites have been successfully constructed, most of them using bottom-up synthetic approaches.

Molecular analysis of the Drosophila miniature-dusky ( m-dy) gene complex: m-dy mRNAs encode transmembrane proteins with similarity to C. elegans cuticulin.

Mutations in the Drosophila miniature-dusky ( m-dy) gene complex were first reported by Morgan and Bridges about 90 years ago. m-dy mutants have abnormally small wings, a phenotype attributed to a cell-autonomous reduction in the size of the epidermal cells comprising the differentiated wing. Using a molecular genetic approach, we have characterized the m-dy chromosomal interval and identified a pair of adjacent transcription units corresponding to m and dy. A dy mutant known as dy (And) has a single base substitution within the protein-coding region that is predicted to result in an amber stop codon and premature translational termination. We show that dy mRNA is expressed at two discrete periods during the life cycle--one during embryonic development and early larval instars, the second during adult development, coincident with wing differentiation. In agreement with the phenotypic similarity of m and dy mutants, sequence comparisons reveal a similarity between the predicted MINIATURE and DUSKY proteins, and indicate that the m and dy genes are members of a larger Drosophila gene family. Both m and dy, as well as other members of this superfamily, are predicted to encode transmembrane proteins with similarity to C. elegans cuticle proteins known as cuticulins. We postulate that m, dy and other members of this protein superfamily function as structural components of the Drosophila cuticulin layer. Such a role for m and dy products in wing differentiation is sufficient to explain the morphological phenotypes associated with m-dy mutants.

Design and Evolution of a Miniature Bcl-2 Binding Protein.

A major challenge for chemical biologists lies in the design of potent and selective ligands for protein surfaces. Here a protein grafting and evolution strategy is used to discover highly potent and specific miniature protein ligands for human Bcl-2 and Bcl-XL . Miniature proteins could be used to dissect, modulate, or analyze a single protein function, irrespective of the other functions the protein may regulate within the proteome.

Methodology for optimizing functional miniature proteins based on avian pancreatic polypeptide using phage display

Synthetic genes for avian pancreatic polypeptide (aPP) and for the miniature DNA binding protein PPBR4 were cloned and expressed on the surface of M13 bacteriophage. We anticipate that these constructs will have utility optimizing the properties of miniature proteins based on aPP that result from our previously described protein grafting procedure.

Highly Specific DNA Recognition by a Designed Miniature Protein

Many DNA-binding proteins employ a short R-helix as their primary DNA recognition element.1-5 When removed from their natural contexts, however, most R-helices neither fold nor bind DNA with high affinity.6,7 Here we describe a strategy for the design of miniature proteins that present a solvent-exposed R-helix able to recognize DNA with high affinity and specificity. This strategy involves dissecting those R-helical residues required for DNA recognition from their native protein context and grafting them on a stable, miniature protein core (Figure 1). This strategy was used to design a 42-amino acid protein displaying the DNAcontact residues of a single basic region-leucine zipper (bZIP) protein. This miniature protein bound DNA in the low nanomolar concentration range under physiological solution conditions and exhibited greater sequence specificity than a natural bZIP protein. This strategy may represent a general approach to the design of small, folded proteins that recognize nucleic acid and protein targets with high affinity and specificity. Our design process began with avian pancreatic polypeptide (aPP), a small, well-folded protein of known structure consisting of a single R-helix stabilized by hydrophobic interaction with a type II polyproline helix (Figure 1).8-10 Since formation of the aPP core requires residues on only one face of the aPP R-helix (shown in blue), the opposite, solvent-exposed face of the R-helix is available for recognition of other macromolecular targets. By grafting various combinations of those thirteen residues (shown in pink) used by GCN4 to recognize the CRE half site (ATGAC, hsCRE)1,2 on the solvent-exposed R-helical face of aPP, we generated a series of polyproline helix-basic region (PPBRSR) molecules containing most or all of the DNA-contact residues of GCN4 and most or all of the folding residues of aPP (Figure 2). This procedure generated three positions of conflict, where essential DNA-contact and aPP-folding residues occupied a single position on the helix (Figure 2). No significant DNA binding was detected with peptides PPBR0SR, PPBR10SR, and PPBR11SR, which lacked one or more of these DNA-contact residues. Highaffinity DNA binding was observed with a peptide that contained these three residues: The equilibrium dissociation constant (Kd) of the PPBR2SR‚hsCRE complex was 5 nM under conditions of physiological ionic strength (Figure 3). DNA affinity was enhanced further by selective alanine substitutions that increased the overall R-helical propensity of the peptide, producing the PPBR4‚hsCRE24 complex whose Kd was 1.5 nM under identical conditions. Formation of the PPBR4‚hsCRE24 complex was unaffected by high concentrations of poly (dIdC)‚(dIdC) or a scrambled CRE site (NON), indicating that the high stability of PPBR4‚hsCRE24 was not due primarily to nonspecific ionic interactions. Circular dichroism experiments indicated that like bZIP peptides,6,11 PPBR4SR attained a fully R-helical conformation only in the presence of specific DNA.12 Although others have described monopartite DNA recognition by basic segment peptides, the affinities reported have been only moderate (60 nM-3 μM), and the complexes are stable only in very low ionic strength buffers.13,14 PPBR4SR represents the first example of high affinity, monopartite, major groove recognition at physiological ionic strength. To examine the contribution of hydrophobic core formation to PPBR4‚hsCRE24 complex stability, we studied peptides G27 * To whom correspondence should be addressed. (1) Ellenberger, T. E.; Brandl, C. J.; Struhl, K.; Harrison, S. C. Cell 1992, 71, 1223. (2) Konig, P.; Richmond, T. J. Mol. Biol. 1993, 233, 139. (3) Aggarwal, A. K.; Rodgers, D. W.; Drottar, M.; Ptashne, M.; Harrison, S. C. Science 1988, 242. (4) Ferre-D’Amare, A. R.; Prendergast, G. C.; Ziff, E. B.; Burley, S. K. Nature (London) 1993, 363, 38. (5) Kissinger, C. R.; Liu, B.; Martin-Blanco, E.; Kornberg, T. B.; Pabo, C. O. Cell 1990, 63, 579. (6) Weiss, M. A.; Ellenberger, T.; Wobbe, C. R.; Lee, J. P.; Harrison, S. C.; Struhl, K. Nature (London) 1990, 347, 575. (7) O’Neil, K. T.; Shuman, J. D.; Ampe, C.; DeGrado, W. F. Biochemistry 1991, 30, 9030. (8) Blundell, T. L.; Pitts, J. E.; Tickle, I. C.; Wood, S. P.; Wu, C.-W. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4175. (9) Tonan, K.; Kawata, Y.; Hamaguchi, K. Biochemistry 1990, 29, 4424. (10) Hermans, J., Jr.; Scheraga, H. A. J. Am. Chem. Soc. 1961, 83, 3283. (11) O’Neil, K. T.; Hoess, R. H.; DeGrado, W. F. Science 1990, 249, 774. (12) The CD spectrum of PPBR4SR was unchanged between 1 and 20 μM, indicating that no detectable changes in secondary structure occurred in this range. Addition of hsCRE DNA significantly increased the R-helix content of PPBR4SR; smaller changes were observed upon addition of hsCEBP DNA (see Supporting Information). (13) Park, C.; Campbell, J. L.; Goddard, W. A., III J. Am. Chem. Soc. 1996, 118, 4235. (14) Morii, T.; Yamane, J.; Aizawa, Y.; Makino, K.; Sugiura, Y. J. Am. Chem. Soc. 1996, 118, 10011. Figure 1. Protein grafting strategy for the design of miniature DNAbinding proteins.

Solvent and solute effects on “inside-outside” conformations and rotation about the peptide bond in a miniature protein model

The physicochemical parameters affecting protein unfolding in relation to peptide bond rotations are briefly reviewed. As a suitable model for the study of solvent and solute effects on amide rotation and inside-outside conformations, the 2,2′-biphenyl analog of N-benzoyl- l-phenylalanine methyl ester (I) was synthesized and resolved enzymatically with α-chymotrypsin. The optically pure substance exists as conformer I a (R,S configuration) with an axial methoxycarbonyl in the crystalline state. Rotation about the biphenyl axis leads to the equatorial conformer I b (S,S configuration) in various solvents. In polar solvents, rotation about the amide is rate limiting. Accurate measurements of this rotation were accomplished by following the rate of change in the maximum amplitude of the biphenyl Cotton band at 256 nm. The high sensitivity of the method allowed rate and equilibrium measurements at 10 −3 M in the absence of intermolecular association. Small differences of the order of 100 cal/mole in ΔG ≠ or ΔG eq could thus be detected accurately. It was found that k obs or k 1 (forward step) for equilibration was linearly related (correlation coefficient of 0.96 for k obs) with E T , the solvent polarity index on Reichardt and Dimroth's scale. Rotation was slowest in water and fastest in carbon tetrachloride, δΔG ≠, being 2.4 kcal/mole. Chaotropic anions, cations, and guanidinium chloride accelerated the rate in water. However, the inside-outside (axial-equatorial; I a I b ) ratio at equilibrium did not correlate in any simple manner with the solvent E T values. Rather, correlation within groups of solvents appeared to exist. It was suggested that solvent association with the amide differs quantitatively in the inside and outside conformations. The position of the equilibrium in water was affected by chaotropic ions but not by urea or quanidinium chloride. Some possible mechanisms are briefly outlined.

Synthesis of secretin. IV. Secondary structure in a miniature protein.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis of secretin. IV. Secondary structure in a miniature proteinAgnes Bodanszky, Miguel A. Ondetti, Viktor Mutt, and Miklos BodanszkyCite this: J. Am. Chem. Soc. 1969, 91, 4, 944–949Publication Date (Print):February 1, 1969Publication History Published online1 May 2002Published inissue 1 February 1969https://doi.org/10.1021/ja01032a026RIGHTS & PERMISSIONSArticle Views80Altmetric-Citations73LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (655 KB) Get e-Alerts Get e-Alerts