Emergence of Cooperative Glucose-Binding Networks in Adaptive Peptide Systems.

Minimalistic peptide-based systems that bind sugars in water are challenging to design due to the weakness of interactions and required cooperative contributions from specific amino-acid side chains. Here, we used a bottom-up approach to create peptide-based adaptive glucose-binding networks by mixing glucose with selected sets of input dipeptides (up to 4) in the presence of an amidase to enable in situ reversible peptide elongation, forming mixtures of up to 16 dynamically interacting tetrapeptides. The choice of input dipeptides was based on amino-acid abundance in glucose-binding sites found in the protein data bank, with side chains that can support hydrogen bonding and CH-π interactions. Tetrapeptide sequence amplification patterns, determined through LC-MS analysis, served as a readout for collective interactions and led to the identification of optimized binding networks. Systematic variation of dipeptide input revealed the emergence of two networks of non-covalent hydrogen bonding and CH-π interactions that can co-exist, are cooperative and context-dependent. A cooperative binding mode was determined by studying the binding of the most amplified tetrapeptide (AWAD) with glucose in isolation. Overall, these results demonstrate that the bottom-up design of complex systems can recreate emergent behaviors driven by covalent and non-covalent self-organization that are not observed in reductionist designs and lead to the identification of system-level cooperative binding motifs.

The Impact of Tyrosine Iodination on the Aggregation and Cleavage Kinetics of MMP-9-Responsive Peptide Sequences.

Matrix metalloproteinase (MMP) enzymes are over-expressed by some metastatic cancers, in which they are responsible for the degradation and remodeling of the extracellular matrix. In recent years, MMPs have emerged as promising targets for enzyme-responsive diagnostic probes because oligopeptides can be designed to be selectively hydrolyzed by exposure to these enzymes. With the ultimate goal of developing radio-iodinated peptides as supramolecular building blocks for MMP-sensitive tools for nuclear imaging and therapy, we designed three MMP-9-responsive peptides containing either tyrosine or iodotyrosine to assess the impact of iodotyrosine introduction to the peptide structure and cleavage kinetics. We found that the peptides containing iodotyrosine underwent more rapid and more complete hydrolysis by MMP-9. While the peptides under investigation were predominantly disordered, it was found that iodination increased the degree of aromatic residue-driven aggregation of the peptides. We determined that these iodination-related trends stem from the improved overall intramolecular order through H- and halogen bonding, in addition to intermolecular organization of the self-assembled peptides due to steric and electrostatic effects introduced by the halogenated tyrosine. These fundamental observations provide insights for the development of enzyme-triggered peptide aggregation tools for localized radioactive iodine-based tumor imaging.

Melanin-Inspired Chromophoric Microparticles Composed of Polymeric Peptide Pigments.

Melanin and related polyphenolic pigments are versatile functional polymers that serve diverse aesthetic and protective roles across the living world. These polymeric pigments continue to inspire the development of adhesive, photonic, electronic and radiation-protective materials and coatings. The properties of these structures are dictated by covalent and non-covalent interactions in ways that, despite progress, are not fully understood. It remains a major challenge to direct oxidative polymerization of their precursors (amino acids, (poly-)phenols, thiols) toward specific structures. By taking advantage of supramolecular pre-organization of tyrosine-tripeptides and reactive sequestering of selected amino acids during enzymatic oxidation, we demonstrate the spontaneous formation of distinct new chromophores with optical properties that are far beyond the range of those found in biological melanins, in terms of color, UV absorbance and fluorescent emission.

Mechanistic insights of evaporation-induced actuation in supramolecular crystals.

Water-responsive materials undergo reversible shape changes upon varying humidity levels. These mechanically robust yet flexible structures can exert substantial forces and hold promise as efficient actuators for energy harvesting, adaptive materials and soft robotics. Here we demonstrate that energy transfer during evaporation-induced actuation of nanoporous tripeptide crystals results from the strengthening of water hydrogen bonding that drives the contraction of the pores. The seamless integration of mobile and structurally bound water inside these pores with a supramolecular network that contains readily deformable aromatic domains translates dehydration-induced mechanical stresses through the crystal lattice, suggesting a general mechanism of efficient water-responsive actuation. The observed strengthening of water bonding complements the accepted understanding of capillary-force-induced reversible contraction for this class of materials. These minimalistic peptide crystals are much simpler in composition compared to natural water-responsive materials, and the insights provided here can be applied more generally for the design of high-energy molecular actuators.

Polymeric peptide pigments with sequence-encoded properties.

Melanins are a family of heterogeneous polymeric pigments that provide ultraviolet (UV) light protection, structural support, coloration, and free radical scavenging. Formed by oxidative oligomerization of catecholic small molecules, the physical properties of melanins are influenced by covalent and noncovalent disorder. We report the use of tyrosine-containing tripeptides as tunable precursors for polymeric pigments. In these structures, phenols are presented in a (supra-)molecular context dictated by the positions of the amino acids in the peptide sequence. Oxidative polymerization can be tuned in a sequence-dependent manner, resulting in peptide sequence-encoded properties such as UV absorbance, morphology, coloration, and electrochemical properties over a considerable range. Short peptides have low barriers to application and can be easily scaled, suggesting near-term applications in cosmetics and biomedicine.

Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations.

The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (<ΦF> = 0.22) that is virtually unaffected by the neighbouring bases (ΦF = 0.20-0.25), resulting in an average brightness of 1900 M-1 cm-1. The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (<ΦF > = 0.24) compared to dsRNA, with a broader distribution (ΦF = 0.17-0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (<ΔT m> = + 2.3 °C). These properties make tCO a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.

A critical base pair in k-turns that confers folding characteristics and correlates with biological function.

Kink turns (k-turns) are widespread elements in RNA that mediate tertiary contacts by kinking the helical axis. We have found that the ability of k-turns to undergo ion-induced folding is conferred by a single base pair that follows the conserved A·G pairs, that is, the 3b·3n position. A Watson-Crick pair leads to an inability to fold in metal ions alone, while 3n=G or 3b=C (but not both) permits folding. Crystallographic study reveals two hydrated metal ions coordinated to O6 of G3n and G2n of Kt-7. Removal of either atom impairs Mg(2+)-induced folding in solution. While SAM-I riboswitches have 3b·3n sequences that would predispose them to ion-induced folding, U4 snRNA are strongly biased to an inability to such folding. Thus riboswitch sequences allow folding to occur independently of protein binding, while U4 should remain unfolded until bound by protein. The empirical rules deduced for k-turn folding have strong predictive value.

Crystal structure and mechanistic investigation of the twister ribozyme.

We present a crystal structure at 2.3-Å resolution of the recently described nucleolytic ribozyme twister. The RNA adopts a previously uncharacterized compact fold based on a double-pseudoknot structure, with the active site at its center. Eight highly conserved nucleobases stabilize the core of the ribozyme through the formation of one Watson-Crick and three noncanonical base pairs, and the highly conserved adenine 3' of the scissile phosphate is bound in the major groove of an adjacent pseudoknot. A strongly conserved guanine nucleobase directs its Watson-Crick edge toward the scissile phosphate in the crystal structure, and mechanistic evidence supports a role for this guanine as either a general base or acid in a concerted, general acid-base-catalyzed cleavage reaction.

Structure and folding of a rare, natural kink turn in RNA with an A*A pair at the 2b*2n position.

The kink turn (k-turn) is a frequently occurring motif, comprising a bulge followed by G•A and A•G pairs that introduces a sharp axial bend in duplex RNA. Natural k-turn sequences exhibit significant departures from the consensus, including the A•G pairs that form critical interactions stabilizing the core of the structure. Kt-23 found in the small ribosomal subunit differs from the consensus in many organisms, particularly in the second A•G pair distal to the bulge (2b•2n). Analysis of many Kt-23 sequences shows that the frequency of occurrence at the 2n position (i.e., on the nonbulged strand, normally G in standard k-turns) is U>C>G>A. Less than 1% of sequences have A at the 2n position, but one such example occurs in Thelohania solenopsae Kt-23. This sequence folds only weakly in the presence of Mg²âº ions but is induced to fold normally by the binding of L7Ae protein. Introduction of this sequence into the SAM-I riboswitch resulted in normal binding of SAM ligand, indicating that tertiary RNA contacts have resulted in k-turn folding. X-ray crystallography shows that the T. solenopsae Kt-23 adopts a standard k-turn geometry, making the key, conserved hydrogen bonds in the core and orienting the 1n (of the bulge-proximal A•G pair) and 2b adenine nucleobases in position facing the opposing minor groove. The 2b and 2n adenine nucleobases are not directly hydrogen bonded, but each makes hydrogen bonds to their opposing strands.

The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether.

Fluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair. Our recent FRET study has suggested that when they are attached via a longer 13-atom tether, these fluorophores are repositioned relative to the terminal basepair by a rotation of ∼30°, while remaining stacked. In this study, we have used NMR to extend our structural understanding to the commonly used fluorophore sulfoindocarbocyanine-3 (sCy3) attached to the 5'-terminus of the double-helical DNA via a 13-atom flexible tether (L13). We find that L13-sCy3 remains predominantly stacked onto the end of the duplex, but adopts a significantly different conformation, from that of either Cy3 or Cy5 attached by 3-atom tethers, with the long axes of the fluorophore and the terminal basepair approximately parallel. This result is in close agreement with our FRET data, supporting the contention that FRET data can be used to provide orientational information.

A structural database for k-turn motifs in RNA.

The kink-turn (k-turn) is a common structural motif in RNA that introduces a tight kink into the helical axis. k-turns play an important architectural role in RNA structures and serve as binding sites for a number of proteins. We have created a database of known and postulated k-turn sequences and three-dimensional (3D) structures, available via the internet. This site provides (1) a database of sequence and structure, as a resource for the RNA community, and (2) a tool to enable the manipulation and comparison of 3D structures where known.