Large Scale Synthesis of Native Turn and Helix Mimics Stabilized by a Generic Hydrogen Bond Surrogate

Short Turn and Helix mimics frequently represent molecular recognition surfaces perturbing bio-relevant protein-biomolecular interfaces. Generic methods that can stabilize short peptides into turns or helices by retaining all recognition elements, have tremendous applications in drug discovery. Here, a versatile modular synthetic protocol is presented for stabilizing turns and helices of different sizes by replacing their ring-closing hydrogen bonds with a generic three-carbon covalent surrogate. Two Fukuyama-Mitsunobu reactions insert the surrogate between desired residues in high yields and purity. Coupling with turn size-dependent oligopeptides containing both sterically restricted and non-coded residues, followed by macrolactamization yield a variety of stabilized turns. Short peptide extensions at the C-terminus of these turns yield stabilized 310-helices and α-helices. This solution-phase synthetic approach provides combinatorial access to libraries of stabilized turns and helices with all their native residues retained, in >100 mmole scales, in about one week per stabilized mimic, to technicians with postgraduate level training.

Biomolecular interfaces based on self-assembly and self-recognition form biosensors capable of recording molecular binding and release

Cellular components manipulated in a synthetic environment form a biosensor capable of evaluating association and dissociation as related to molecular self-recognition and self-assembly.

Local dynamics of proteins and DNA evaluated from crystallographicBfactors

The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-calledBfactors. To find out how the crystal-derivedBfactors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaledBfactors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneideret al.(2014),Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e.forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. TheBfactors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids haveBfactors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of theBfactors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are surprising. The features discriminating different types of residues are less pronounced in structures with lower crystallographic resolution. Some of the observed trends are likely to be the consequence of improper refinement protocols that may need to be rectified.

Local dynamics of proteins and DNA evaluated from crystallographic B-factors

Temperature displacement factors also known as B-factors provide information about local dynamics of atoms. Because dynamic behavior of biomolecules is as important as their static molecular structures we decided to analyze B-factors in almost a thousand non-redundant crystal structures of protein-DNA complexes from a well-curated dataset described in Schneider et al. Nucleic Acids Research, 42 (2014). Biopolymer residues, amino acids and nucleotides, were classified according to their molecular neighborhood as solvent-accessible, solvent-inaccessible (buried, i. e. forming the protein core), or lying at protein-protein or protein-DNA interfaces. In addition, water molecules were labeled as solely bound to the biopolymer surface as the first hydration shell or as water bridges binding protein and DNA molecules. Distributions of scaled B-factors for these types of residues confirmed several expected features of protein and DNA dynamics but they also revealed some surprising facts. Solvent-accessible amino acids have B-factors larger than residues at both biomolecular interfaces and amino acids forming protein core are restricted in their movement the most. A really unique feature of the buried amino acids is that their side chains are restricted in their movements more than the main chains. Protein interior is therefore packed significantly better than protein-protein or protein-DNA interfaces. Low values of B-factors of the waters bridging protein and DNA molecules contrast with extremely high values of DNA phosphates. Characteristic features distinguishing different types of residues quickly vanish in structures with lower resolution and some of the observed trends are a likely consequence of improper refinement protocols that may need rectifying. Acknowledgments. This study was supported by BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF and by grant P305/12/1801 from the Czech Science Foundation.