Folding Preferences Research Articles

Solvent-Dependent Folding Behavior of a Helix-Forming Aryl-Triazole Foldamer.

Aromatic foldamers make up a novel class of bioinspired molecules that display helical conformations and have functions that rely on control over their coil-helix folding preferences. While the folding has been extensively examined by experiment, it has rarely been paired with the types of atomic level insights offered by theory. We present the results of all-atom molecular dynamics (MD) simulations to examine the role of solvent polarity on driving the helical folding behavior of the aryl-triazole foldamer. The temperature-dependent enhanced sampling technique, replica-exchange MD simulations, was employed to understand the folding phenomena. The simulation results show that in a low polarity solvent (dichloromethane), the foldamer prefers to stay in the unfolded state. The unfolded state has four dipolar triazoles (5 D) in their favored all-anti geometries and favoring anti-parallel geometries. However, an increase in solvent polarity using acetonitrile resulted in solvophobic collapse, yielding the helically folded form as the predominant state. The folded helix has an all-syn geometry, with triazoles in parallel arrangements. Intermediate conformations with a mixture of syn and anti arrangements of the triazoles are of lower abundance in both the DCM and MeCN solvents. The chiral handedness of the helix observed experimentally is assigned as left-handed by correlation with computed electronic circular dichroism spectra using time-dependent density functional theory.

Effects of chirality and side chain length in Cα,α-dialkylated residues on β-hairpin peptide folded structure and stability.

Strategic incorporation of achiral Cα,α-dialkylated amino acids with bulky substituents into peptides can be used to promote extended strand conformations and inhibit protein-protein interactions associated with amyloid formation. In this work, we evaluate the thermodynamic impact of chiral Cα,α monomers on folding preferences in such systems through introduction of a series of Cα-methylated and Cα-ethylated residues into a β-hairpin host sequence. Depending on stereochemical configuration of the artificial monomer and potential for additional hydrophobic packing, a Cα-ethyl-Cα-propyl glycine residue can provide similar or enhanced folded stability relative to an achiral Cα,α-diethyl analogue.

Structural Water Stabilizes Protein Motifs in Liquid Protein Phase: The Folding Mechanism of Short β-Sheets Coupled to Phase Transition.

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small β-sheets, the key motifs of proteins, undergoing structural changes in liquid–liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3–0.5 kcal·mol−1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2–2.3 kcal·mol−1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol−1 per residue excess stabilization.

Synthesis and Insecticidal Evaluation of Novel Anthranilic Diamides Derivatives Containing 4‐Chlorine Substituted N‐Pyridylpyrazole

Main observation and conclusionTo search for potent insecticides targeting at ryanodine receptors (RyRs), a series of novel anthranilic diamides analogs containing 4‐chlorine N‐pyridylpyrazole were designed and synthesized. Their insecticidal activities were evaluated and the preliminary structure‐activity relationships (SARs) were discussed. The insecticidal results showed that some of the compounds (8a—8h, 8m, 8n) exhibited good larvicidal activities against oriental armyworm at 2.5 mg·L–1, and compound 8m possessed 60% insecticidal activity at 0.5 mg·L–1. For diamondback moth, 8m exhibited better activity than Chlorantraniliprole at a hundred fold preference. The calcium imaging technique experiment results suggested that compound 8m could increase the intracellular [Ca2+]i. With the neurons preincubated experiment, the results confirmed that the target of this series of compounds could be RyRs in the central larvae neurons of oriental armyworm. The results indicated that compound 8m could respond as a potential modulator of the insect ryanodine receptor.

The fold preference and thermodynamic stability of α-synuclein fibrils is encoded in the non-amyloid-β component region.

The heterogeneity of the synucleinopathies, neurological disorders that include Parkinson's disease (PD), indicates that toxicity, seeding/cross-seeding ability, and propagation of α-synuclein (αS) assemblies depend on their distinct structural characteristics or "strain". To examine the molecular signature that encodes the aggregation seed, conformational preference, and thermodynamic stability of full-length αS fibrils, we performed molecular dynamics simulations on two non-amyloid-β component (NAC) fibril structures, containing residues 61-95 of two distinct αS fibrils. We identified several discrete hot spots in the recognized hydrophobic core of NAC (residues 68-82) that could initiate the early assembly of αS. We show that NAC fibrils inherit the preferred fold of their parent αS fibril, but could switch conformational preference in two fibril mutants K80Q and E83Q under different solution conditions. Similar to αS fibrils, NAC fibrils are also sensitive to temperature and salt concentration. The favorable solvation free energy of NAC fibrils at low temperature (280 K) suggests a propensity for cold-denaturation. Our results indicate that the strain-dependent synucleinopathies may be partially imprinted in the fold-dependent thermodynamic properties of NAC fibrils, providing structural insights into the emerging development of anti-PD treatments that target the NAC region of αS.

The role of weak interactions in characterizing peptide folding preferences using a QTAIM interpretation of the Ramachandran plot (ϕ‐ψ)

AbstractThe original Ramachandran plot is a potent way to understand structures of biomolecules, however only backbone conformations are considered. We formulate a new interpretation of the original Ramachandran plot (ϕ‐ψ) that can include a description of the weaker interactions including both the hydrogen bonds and HH bonds as a new way to derive insights into the phenomenon of peptide folding. Specifically, we show that QTAIM analysis permits identifying key regions of the Ramachandran plot without the need for massive data sets. The QTAIM interpreted Ramachandran plot is derived from QTAIM eigenvectors and not a trivial coordinate transformation. An investigation of both the backbone and the weaker bonds within the framework of the QTAIM interpreted Ramachandran plot was found to be in line with physical intuition. The least‐preferred directions calculated for the hydrogen bonds and HH bonds were found to coincide with the “unlikely” regions of the Ramachandran plot.

IGF1 and IGF2 specificities to the two insulin receptor isoforms are determined by insulin receptor amino acid 718.

MethodsAlanine scan of insulin receptor (IR)-B exon 11 and site-directed mutagenesis of amino acid 718 in human IR-A and IR-B were performed. Ligand affinities to wild type and mutated receptors were studied by displacement of radioactive insulin in binding assay on secreted soluble midi receptors or solubilized semi-purified full length receptors stably expressed in Baby Hamster Kidney cells. Phosphorylation of IR in response to insulin, IGF1 and IGF2 was measured using ELISA.ResultsInsulin, insulin detemir and insulin glargine maximally showed two fold differences in affinity for human IR-A and IR-B, but IGF1 and IGF2 had up to 10 fold preference for IR-A. Alanine scan of exon 11 revealed that position 718 is important for low IGF1 affinity to IR-B. Mutational analysis of amino acid residue 718 in IR-A and IR-B demonstrated that charge is important for IGF1 and IGF2 affinity but not important for insulin affinity. The affinity of IGF1 and IGF2 for the mutant IR-A P718K was comparable to the wild type IR-B whereas the affinity of IGF1 and IGF2 for the mutant IR-B K718P was comparable to the wild type IR-A. Changes in affinity were also reflected in the IR activation pattern.ConclusionMutating position 718 in human IR-B to the proline found at position 718 in human IR-A increased IGF1 and IGF2 affinity to a level comparable to IR-A and mutating position 718 in IR-A to the lysine found at position 718 in IR-B decreased IGF1 and IGF2 affinity to a level comparable to IR-B, whereas a negatively charged glutamate did not. These changes in the affinities were also reflected in the IR phosphorylation pattern, meaning that position 718 is important for both affinity and activation of the receptor. It should be emphasized that none of the mutations affected insulin affinity, indicating that the mutations did not alter the overall receptor structure and that the effect is ligand specific.

Alternative DNA structure formation in the mutagenic human c-MYC promoter.

Mutation ‘hotspot’ regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene.

Theoretical Insights into the Biophysics of Protein Bi-stability and Evolutionary Switches.

Deciphering the effects of nonsynonymous mutations on protein structure is central to many areas of biomedical research and is of fundamental importance to the study of molecular evolution. Much of the investigation of protein evolution has focused on mutations that leave a protein’s folded structure essentially unchanged. However, to evolve novel folds of proteins, mutations that lead to large conformational modifications have to be involved. Unraveling the basic biophysics of such mutations is a challenge to theory, especially when only one or two amino acid substitutions cause a large-scale conformational switch. Among the few such mutational switches identified experimentally, the one between the GA all-α and GB α+β folds is extensively characterized; but all-atom simulations using fully transferrable potentials have not been able to account for this striking switching behavior. Here we introduce an explicit-chain model that combines structure-based native biases for multiple alternative structures with a general physical atomic force field, and apply this construct to twelve mutants spanning the sequence variation between GA and GB. In agreement with experiment, we observe conformational switching from GA to GB upon a single L45Y substitution in the GA98 mutant. In line with the latent evolutionary potential concept, our model shows a gradual sequence-dependent change in fold preference in the mutants before this switch. Our analysis also indicates that a sharp GA/GB switch may arise from the orientation dependence of aromatic π-interactions. These findings provide physical insights toward rationalizing, predicting and designing evolutionary conformational switches.

Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

Mimicking the First Turn of an α-Helix with an Unnatural Backbone: Conformation-Specific IR and UV Spectroscopy of Cyclically Constrained β/γ-Peptides

The folding preferences of two capped, constrained β/γ-dipeptide isomers, Ac-βACPC-γACHC-NHBn and Ac-γACHC-βACPC-NHBn, (designated βγ and γβ, respectively), have been investigated using single- and double-resonance ultraviolet and infrared spectroscopy in the gas phase. These capped β/γ-dipeptides have the same number of backbone atoms between their N- and C-termini as a capped α-tripeptide and thus serve as a minimal structural unit on which to test their ability to mimic the formation of the first turn of an α-helix. Resonant two-photon ionization and UV-UV hole-burning spectroscopy were performed in the S0-S1 region, revealing the presence of three unique conformations of βγ and a single conformation of γβ. Resonant ion-dip infrared spectra were obtained in the NH stretch region from 3300 to 3500 cm(-1) and in both the amide I and amide II regions from 1400 to 1800 cm(-1). These infrared spectra were compared to computational predictions from density functional theory calculations at the M05-2X/6-31+G(d) level, leading to assignments for the observed conformations. Two unique bifurcated C8/C13 H-bonded ring structures for βγ and a single bifurcated C9/C13 H-bonded ring structure for γβ were observed. In all cases, the H-bonding patterns faithfully mimic the first full turn of an α-helix, most notably by containing a 13-membered H-bonded cycle but also by orienting the interior amide group so that it is poised to engage in a second C13 H-bond as the β/γ-peptide lengthens in size. The structural characteristics of the β/γ-peptide version of the 13-helix turn are compared with the α-helix counterpart and with a reported crystal structure for a longer β/γ-peptide oligomer.

Structural characterization of short hybrid urea/carbamate (U/C) foldamers: A case of partial helix unwinding

Aliphatic oligoureas−(NH−CH(R)−CH2−NH−CO)n− and oligocarbamates −(NH−CH(R)−CH2−O−CO)n− are two classes of synthetic peptidomimetic oligomers whose backbone is isosteric to that of γ-peptides. We have shown recently that the constituent units of these backbones (i.e., amide (A), carbamate (C) and urea (U) units) can be combined in various ways to generate new heterogeneous oligomers with well-defined secondary structures. For example, oligomers consisting of urea (U) and carbamate (C) linkages arranged in a 1:1 pattern adopt a helical conformation akin to that of urea homoligomers and γ-peptide foldamers. In this case, helix formation is mainly driven by U units whose propensity for folding surpasses that of C units. Here, we have investigated further the influence of the U/C ratio on the folding preference of such heterogeneous oligomers. We report the synthesis and the structural analysis of a short oligomer with a 2:3 U/C ratio and two consecutive carbamate linkages. X-ray diffraction analysis reveals a helical structure that unwinds at one end. In contrast, a cognate oligomer prepared for comparison and containing four contiguous urea units adopts a fully helical conformation in the crystalline state. These results which are supported by data in solution indicate that the balance between U and C units should be carefully adjusted and that consecutive C linkages should be avoided for optimal helix formation.

Foldamers of β-peptides: conformational preference of peptides formed by rigid building blocks. The first MI-IR spectra of a triamide nanosystem

To determine local chirality driven conformational preferences of small aminocyclobutane-1-carboxylic acid derivatives, X-(ACBA) n -Y, their matrix-isolation IR spectra were recorded and analyzed. For the very first time model systems of this kind were deposited in a frozen (~10K) noble gas matrix to reduce line width and thus, the recorded sharp vibrational lines were analyzed in details. For cis-(S,R)-1 monomer two "zigzag" conformers composed of either a six or an eight-membered H-bonded pseudo ring was identified. For trans-(S,S)-2 stereoisomer a zigzag of an eight-membered pseudo ring and a helical building unit were determined. Both findings are fully consistent with our computational results, even though the relative conformational ratios were found to vary with respect to measurements. For the dimers (S,R,S,S)-3 and (S,S,S,R)-4 as many as four different cis,trans and three different trans,cis conformers were localized in their matrix-isolation IR (MI-IR) spectra. These foldamers not only agree with the previous computational and NMR results, but also unambiguously show for the first time the presence of a structure made of a cis,trans conformer which links a "zigzag" and a helical foldamer via a bifurcated H-bond. The present work underlines the importance of MI-IR spectroscopy, applied for the first time for triamides to analyze the conformational pool of small biomolecules. We have shown that the local chirality of a β-amino acid can fully control its backbone folding preferences. Unlike proteogenic α-peptides, β- and especially (ACBA) n type oligopeptides could thus be used to rationally design and influence foldamer's structural preferences.

Helix compactness and stability: Electron structure calculations of conformer dependent thermodynamic functions

Structure, stability, cooperativity and molecular packing of two major backbone forms: 310-helix and β-strand are investigated. Long models HCO-(Xxx)n-NH2 Xxx=Gly and (l-)Ala, n⩽34, are studied at two levels of theory including the effect of dispersion forces. Structure and folding preferences are established, the length modulated cooperativity and side-chain determined fold compactness is quantified. By monitoring ΔG°β→α rather than the electronic energy, ΔEβ→α, it appears that Ala is a much better helix forming residue than Gly. The achiral Gly forms a more compact 310-helix than any chiral amino acid residue probed here for l-Ala.

Exploiting diverse stereochemistry of β-amino acids: toward a rational design of sheet-forming β-peptide systems

Due to the two methylene groups in their backbone, β-amino acids can adopt numerous secondary structures, including helices, sheets and nanotubes. Chirality introduced by the additional side chains can significantly influence the folding preference of β-peptides composed of chiral β-amino acids. However, only conceptual suggestions are present in the literature about the effect of chirality on folding preferences. Summarizing both the experimental and computational results, Seebach (Chem Biodivers 1:1111-1240, 2004) has proposed the first selection rule on the effect of side chain chirality, on the folding preference of β-peptides. In order to extend and fine-tune the aforementioned predictions of Seebach, we have investigated its validity to the novel type of apolar sheet proposed recently (Pohl et al. in J Phys Chem B 114:9338-9348, 2010). In order to facilitate the rational design of sheet-like structures, a systematic study on the effect of chirality on "apolar" sheet stability is presented on disubstituted [HCO-β-Ala-β(2,3)-hAla-β-Ala-NH(2)](2) model peptides calculated at the M05-2X/6-311++G(d,p)//M05-2X/6-31G(d) and B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) levels of theory both in vacuum and in polar and apolar solvents. In addition, both types of "apolar" sheets were investigated; the one with two strands of identical (AA) and enantiomeric (AB) backbone structure. Our results show that heterochirally disubstituted sheets have the greatest preference for sheet formation (ΔG ~ -11 kcal mol(-1)). However, in contrast to Seebach's predictions, "homochiral disubstitution" itself does not necessarily disrupt the sheet structure, rather it could result stable fold (ΔG ~ -5 kcal mol(-1)). Results indicate that both the methyl group orientation and the local conformational effect of substitution affects sheet stability, as point chirality was found to have influence only on the backbone torsional angles. These results enabled us to extend and generalize Seebach's predictions and to propose a more general and accurate "rule of thumb" describing the effect of chirality on sheet stability. This offers an easy-to-use summary on how to design β-peptide sheet structures. We conclude that heterochirally disubstituted models are the best candidates for sheet formation, if the two strands are substituted in a way to create identical torsional angle sets on the two backbones for ideal hydrogen-bonding pattern. With adequately selected side chains, homochirally disubtituted derivatives may also form sheet structures, and the position of methyl groups would prevent assembly of more than two strands making it ideal to create hairpins.

Library synthesis and cytotoxicity of a family of 2-phenylacrylonitriles and discovery of an estrogen dependent breast cancer lead compound

In our efforts to prevent highly toxic compounds progressing through our anti-parasitic drug development program, we serendipitously discovered a family of 2-phenylacrylonitriles with excellent growth inhibition of a panel of ten human cancer cell lines. Focused library approaches facilitated the identification of a simple pharmacophore, comprising two terminal aromatic moieties linked via a conjugated cyano (acrylonitrile) moiety. Efforts that perturbed this pharmacophore resulted in a significant drop in growth inhibition. Multiple libraries led to the discovery of two key lead compounds. The first, (Z)-2-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)acrylonitrile (31) exhibits broad spectrum growth inhibition with GI50 values of 0.52–3 μM (HT29 and BE2-C cancer cell lines respectively; average = 1.6 μM). Of greater note is (Z)-2-(3,4-dichlorophenyl)-3-(4-nitrophenyl)acrylonitrile (28), a 0.127 ± 0.043 μM growth inhibitor of the estrogen receptor positive (ER+ve) human breast cancer cell line, MCF-7. Analogue 28 displays up to 543 fold selectivity towards MCF-7 cells compared with nine other non-breast derived cancer cell lines. Further screening of 28 against one human, ER−ve breast cancer cell line (MDA-MB231) and one normal non-tumourigenic breast epithelial cell line (MCF-10A) returned poor growth inhibition values of 34 ± 2 and 16 ± 4μM, demonstrating ca. ∼268 and∼126 fold preference for the MCF-7 estrogen dependent breast cancer cells.

Kinetics and Docking Studies of a COX-2 Inhibitor Isolated from Terminalia bellerica Fruits

Triphala is an Ayurvedic herbal formulation consisting of equal parts of three myrobalans: Terminalia chebula, Terminalia bellerica and Emblica officinalis. We recently reported that chebulagic acid (CA) isolated from Terminalia chebula is a potent COX-2/5-LOX dual inhibitor. In this study, compounds isolated from Terminalia bellerica were tested for inhibition against COX and 5-LOX. One of the fractionated compounds showed potent inhibition against COX enzymes with no inhibition against 5-LOX. It was identified as gallic acid (GA) by LC-MS, NMR and IR analyses. We report here the inhibitory effects of GA, with an IC(50) value of 74 nM against COX-2 and 1500 nM for COX-1, showing ≈20 fold preference towards COX-2. Further docking studies revealed that GA binds in the active site of COX-2 at the non-steroidal anti-inflammatory drug (NSAID) binding site. The carboxylate moiety of GA interacts with Arg120 and Glu524. Based on substrate dependent kinetics, GA was found to be a competitive inhibitor of both COX-1 and COX-2, with more affinity towards COX-2. Taken together, our studies indicate that GA is a selective inhibitor of COX-2. Being a small natural product with selective and reversible inhibition of COX-2, GA would form a lead molecule for developing potent anti-inflammatory drug candidates.

Identification of folding preferences of cleavage junctions of HIV-1 precursor proteins for regulation of cleavability

Human immunodeficiency virus type 1 protease (HIV-1 PR) cleaves two viral precursor proteins, Gag and Gag-Pol, at multiple sites. Although the processing proceeds in the rank order to assure effective viral replication, the molecular mechanisms by which the order is regulated are not fully understood. In this study, we used bioinformatics approaches to examine whether the folding preferences of the cleavage junctions influence their cleavabilities by HIV-1 PR. The folding of the eight-amino-acid peptides corresponding to the seven cleavage junctions of the HIV-1(HXB2) Gag and Gag-Pol precursors were simulated in the PR-free and PR-bound states with molecular dynamics and homology modeling methods, and the relationships between the folding parameters and the reported kinetic parameters of the HIV-1(HXB2) peptides were analyzed. We found that a folding preference for forming a dihedral angle of Cβ (P1)-Cα (P1)- Cα (P1')-Cβ (P1') in the range of 150 to 180 degrees in the PR-free state was positively correlated with the 1/K(m) (R = 0.95, P = 0.0008) and that the dihedral angle of the O (P2)-C (P2)- C (P1)- O (P1) of the main chains in the PR-bound state was negatively correlated with k(cat) (R = 0.94, P = 0.001). We further found that these two folding properties influenced the overall cleavability of the precursor protein when the sizes of the side chains at the P1 site were similar. These data suggest that the dihedral angles at the specific positions around the cleavage junctions before and after binding to PR are both critical for regulating the cleavability of precursor proteins by HIV-1 PR.

Natural and artificial RNAs occupy the same restricted region of sequence space

Different chemical and mutational processes within genomes give rise to sequences with different compositions and perhaps different capacities for evolution. The evolution of functional RNAs may occur on a "neutral network" in which sequences with any given function can easily mutate to sequences with any other. This neutral network hypothesis is more likely if there is a particular region of composition that contains sequences that are functional in general, and if many different functions are possible within this preferred region of composition. We show that sequence preferences in active sites recovered by in vitro selection combine with biophysical folding rules to support the neutral network hypothesis. These simple active-site specifications and folding preferences obtained by artificial selection experiments recapture the previously observed purine bias and specific spread along the GC axis of naturally occurring aptamers and ribozymes isolated from organisms, although other types of RNAs, such as miRNA precursors and spliceosomal RNAs, that act primarily through complementarity to other amino acids do not share these preferences. These universal evolved sequence features are therefore intrinsic in RNA molecules that bind small-molecule targets or catalyze reactions.

Conformationally Constrained Mimetics of Laminin Peptide YIGSR as Precursors for Antimetastatic Disintegrins

Conformationally constrained mimetics of the laminin cell-adhesion site, YIGSR, are described. The site is the natural antagonist of the integrin-associated laminin receptor 1 (LAMR1) known to mediate metastatic tumor adhesion. The attachment of selected metastatic cell lines toward the constrained antagonists has been assessed. Observed differential responses prompted by folding preferences of the mimetics revealed stronger attachment activities for turnlike structures. The results permit the conformational design of antimetastatic disintegrins.