Marcey was the section editor for the chemical biology section of the new reference work Supramolecular Chemistry: From Molecules to Nanomaterials.
Published in JACS, researchers in the Papanikolas and Waters groups, in collaboration with members of the Meyer group at Carolina Chemistry and the Papoian Group at the University of Maryland, describe how solid-phase peptide synthesis has been applied to the preparation of phosphonate-derivatized oligoproline assemblies containing two different RuII polypyridyl chromophores coupled via "click" chemistry.
In water or methanol the assembly adopts the polyproline II (PPII) helical structure, which brings the chromophores into close contact. Excitation of the assembly is followed by rapid, efficient intra-assembly energy transfer to the inner RuII. The oligoproline/click chemistry approach holds great promise for the preparation of interfacial assemblies for energy conversion based on a family of assemblies having controlled compositions and distances between key functional groups.
Waters Receives Keck Grant
Professor Marcey Waters will be the Primary Investigator for a $1 million grant from the W.M. Keck Foundation's Medical Research Program to study a widespread but largely unexplored phenomenon that may be implicated in many diseases, including cancer. The phenomenon, called protein methylation, has added a new dimension in our understanding of how genes and other aspects of the cell are regulated, explains Professor Waters. Proteins are modified with these chemical tags, which in turn change their behavior in ways that are important for turning on or off their functions.
The research aims to provide researchers with a map of these chemical tags and the patterns with which these tags decorate the surface of different proteins. A visual and/or chemical representation of these patterns may provide breakthrough insights into why certain cells become diseased while others stay healthy. Furthermore, Waters and her co-principal investigators, Brian D. Strahl and Xian Chen, associate professors in the department of biochemistry and biophysics in UNC's School of Medicine, plan to use these new tools to pinpoint precisely which molecular interactions within cells break down and lead to disease. This could open the door to the development of highly specific and targeted therapies.
Members of the Waters Group, published in Angewandte Chemie, International Edition, report on the study of a minimal mimic of a protein domain that binds to type II polyproline helices through an aromatic cleft.
This binding motif mimics that of protein domains, including those important in disease states such as HIV infection and cancer. The study provides insight into the structure–function relationship in binding as well as quantitative data on the magnitude of prolyl–π interactions relevant to inhibitor design.
Waters Named WOWS Scholar
Carolina Chemistry Professor Marcey Waters has been named 2011-2013 WOWS, Working on Women in Science, Scholar for the College of Arts and Sciences. The honor recognizes her role as an outstanding scholar, teacher, mentor and leader, and will support her activities over the next two years to advance the status of women in the sciences at Carolina.
"As exceptional scholar, teacher and mentor, Professor Waters have also been an exceptional advocate for women in the science," said Karen M. Gil, Dean of the College. "I am confident she will make important new contributions to enhance the advancement of women scientists on our campus in her role as WOWS Scholar."
Professor Waters specializes in bioorganic chemistry and molecular recognition. Her interdisciplinary research has potential applications in drug delivery, protein design and bio-sensing. She also has mentored women at the undergraduate, graduate and postdoctoral level and has worked to involve more female colleagues in science lecture series and conference presentations. She also has enhanced the process for recruiting women faculty through her service on numerous search committees.
Dueling Post-Translational Modifications
Protein post-translational modifications, PTMs, are used in nature as a means of turning on or off a myriad of biological events. Methylation of lysine and phosphorylation of serine are important PTMs in the histone code found to modulate chromatin packing, which in turn affects gene expression. The design of peptides that fold into secondary structures can help to further our understanding of complex protein interactions.
The Waters Group, published in JACS, reports the design of the Trpswitch peptide sequence that folds into a moderately stable β-hairpin structure in aqueous solution and show that the stability of the structure can be tuned by incorporation of dimethyllysine or phosphoserine. Dimethylated Trpswitch results in an increase in β-hairpin stability, while phosphorylated Trpswitch is unstructured at neutral pH. When both modifications are incorporated into Trpswitch, a less stable β-hairpin structure is observed. This system provides a model to demonstrate how multiple PTMs may work in concert or against each other to influence structure.
NSF Graduate Research Fellowship
Joshua Beaver, a graduate student in the Waters Group, has been selected to receive a National Science Foundation Graduate Research Fellowship. Joshua was selected based on his outstanding abilities and accomplishments, as well as his potential to contribute to strengthening the vitality of the U.S. science and engineering enterprise. The fellowship award will provide Joshua with research support over a five year period.
Joshua's research focuses on using dynamic combinatorial chemistry, DCC, to identify novel host-guest assemblies for the quantification and detection of 3-nitrotyrosine, 3-NT, a post-translational modification found on proteins. Elevated levels of 3-NT is proposed to affect protein function and may be a predictor of disease. The low prevalence of 3-NT in the body and the reactivity of tyrosine in the extreme environments of current analytical methods hinders our ability to adequately detect, isolate, or amplify modified proteins without altering their activity or biasing results. Joshua intends to use DCC to synthesize small-molecule macrocycles that mimic protein binding pockets to selectively bind 3-NT modifications. Ultimately, selective binding under physiological conditions could result in use of these inexpensive, small-molecule receptors instead of antibodies as biomolecular recognition agents for the in vivo analysis of post-translational modifications and other biologically interesting targets.
Understanding Post-Translational Modifications
Researchers in the Waters Group, demonstrate in an article published in ChemComm, dynamic combinatorial chemistry's power for developing synthetic hosts for biological applications. A small molecule receptor that mimics the HP1 chromodomain's affinity for trimethyl lysine has been identified from a dynamic combinatorial library.
Discrimination for trimethyl lysine over the lower methylation states parallels that of the native protein receptor. These studies demonstrate the feasibility of small molecule receptors as potential sensors for protein post-translational modifications.
Thermally Stable β-Hairpin Peptides
Research recently published in Biochemistry by the Waters Group shows how two tryptophan residues were incorporated on one face of a β-hairpin peptide to form an aromatic pocket that interacts with a lysine or N-methylated lysine via cation−π interactions. The two tryptophan residues were found to pack against the lysine side chain forming an aromatic pocket similar to those observed in trimethylated lysine receptor proteins.
Thermal analysis of methylated lysine variant hairpin peptides revealed an increase in thermal stability as the degree of methylation was increased, resulting in the most thermally stable β-hairpin reported to date.
Photoswitchable Dynamic Libraries
Dynamic combinatorial chemistry is a method for discovery of complex host systems from simple building blocks using reversible linkages under thermodynamic control. The Waters group has reported "doubly dynamic" libraries through the use of a novel azobenzene containing monomer which can undergo two reversible processes, hydrazone exchange and photoisomerization, allowing for the development of photoswitchable receptors, published in J. Org Chem.
Libraries which were generated under thermal conditions were dominated by trans isomers of the azobenzene macrocycles, whereas light-induced isomerization resulted in a conformational change of the library members to their corresponding cis-azo form. In the presence of a pentaproline template stabilization and amplification of a specific cis azobenzene host macrocycle was observed, indicative of favorable binding interactions in this host-guest system. The trans-isomer was not amplified, indicating selective binding in the cis-conformation. This system provides a promising new route for discovering light-activated receptors.
Investigation of Carbohydrate-π Interactions
The Waters group recently reported the characterization of a carbohydrate-π interaction, which is implicated in carbohydrate recognition by a wide range of proteins.
This work was published in Chemical Communications and highlighted as a "hot article". Understanding the energetics and driving force of this interaction could lead to new approaches for disrupting carbohydate-protein interactions, which mediate a wide range of biological events, including cell-cell communication and bacterial and viral infection.
Researchers in the Waters Group, as described in an article published in JACS, utilized dynamic combinatorial chemistry to identify a novel small molecule receptor, A2D, for asymmetric dimethyl arginine, aRMe2, which is a post-translational modification, PTM, in proteins. It is known to play a role in a number of diseases, including spinal muscular atrophy, leukemia, lymphoma, and breast cancer.
The receptor exhibits 2.5–7.5-fold selectivity over the isomeric symmetric dimethyl arginine, depending on the surrounding sequence, with binding affinities in the low micromolar range. The affinity and selectivity of A2D for the different methylated states of Arg parallels that of proteins that bind to these PTMs. Characterization of the receptor–PTM complex indicates that cation−π interactions provide the main driving force for binding, loosely mimicking the binding mode found in the recognition of dimethyl arginine by native protein receptors.
Tunable Energy Transfer Rates
Published in Inorganic Chemistry, a collaboration between the Waters and Papanikolas groups outlines energy transfer studies in a series of Ru(II) and Os(II) linked coiled-coil peptides in which the supramolecular scaffold controls the functional properties of the assembly. The researchers describe a general and convergent method for the site-specific incorporation of bipyridyl Ru(II) and Os(II) complexes using solid-phase peptide synthesis and the copper-catalyzed azide–alkyne cycloaddition. Supramolecular assembly positions the chromophores for energy transfer.
Using time-resolved emission spectroscopy the groups measured position-dependent energy transfer that can be varied through changes in the sequence of the peptide scaffold. High level molecular dynamics simulations were used in conjunction with the spectroscopic techniques to gain molecular-level insight into the observed trends in energy transfer. The most efficient pair of Ru(II) and Os(II) linked peptides as predicted by molecular modeling also exhibited the fastest rate of energy transfer. Additionally, the emission quenching for the Ru(II) and Os(II) peptides can be fit to binding models that agree with the dissociation constants determined for the peptides via chemical denaturation.
Tuning HP1α Chromodomain Selectivity
Histone lysine methylation is a critical marker for controlling gene expression. The position and extent of methylation controls the binding of effector proteins that determine whether the associated DNA is expressed or not. Dysregulation of histone protein methylation has been associated with a number of types of cancer, and development of inhibitors for the effector proteins is becoming an active area of research.
Mutation studies performed by scientists in the Waters Group, published in ChemBioChem, provide insight into the role of electrostatic interactions and hydrogen bonding in the differentiation of methylation states and have implications regarding the evolutionary pressure for selectivity in this protein–protein interaction. Moreover, the information from this study may help guide inhibitor development for this class of proteins.
Researchers in the Waters Group have redesigned a β-sheet miniprotein based on the FBP11 WW1 domain sequence for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)2, binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. Subsequent results, published in Biochemistry, extend those studies to a three-stranded β-sheet miniprotein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA.
The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well-structured β-hairpin made of strands 2 and 3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein−DNA interactions as well as a novel example of protein redesign.
Dynamic Cyclic Thiodepsipeptide Libraries
A collaborative effort between the Gagné Group, the Waters Group, and the U.S. Army Research Office, published in Organic Letters, describes how Thiol−thioester exchange was found to readily generate libraries of cyclic thiodepsipeptides under thermodynamic control, which will enable their use in a variety of dynamic combinatorial chemistry assays.
The article discusses the kinetic determinants of macrocycle formation and the role of amino acid structure on the reaction dynamics.
Multiple Myeloma Research
An NIH Research Project Grant, which will fund an interdepartmental collaborative investigation into Multiple Myeloma, has been awarded to Carolina Chemistry Professors Nancy Allbritton, MD, PhD, and Marcey Waters, PhD, and Assistant Professor Peter Voorhees, MD, from UNC's Lineberger Comprehensive Cancer Center.
Multiple Myeloma is a disease with a very poor survival rate and the second most common hematologic malignancy in the United States, where advances in therapy are being aggressively sought. While the disease remains incurable, there are widespread translational efforts to improve chemotherapeutic options in this disease. Molecularly targeted therapies, particularly those directed at the proteasome, but more recently kinase targets as well, are showing clinical efficacy.
The grant was awarded by NIH to this interdisciplinary collaboration with the aim at creating the analytical and chemical tools needed to directly measure the enzymatic activities of protein kinases and the proteasome in cells taken directly from patients with multiple myeloma. The investigators will develop unique fluorescent reagents to report the activity of the protein targets of molecular-based therapies currently in use or in clinical trials. Kinase substrates will be modified to create long- lived compounds by attachment of stably folded beta-hairpin structures or "protectides." The goal of the investigation is to develop an assay to study active signal transduction pathways in the tumor cells of patients, which would enhance diagnostics and prognostics as well aid in therapeutic planning.
Controlling Peptide Folding
Researchers in the Waters Group, as published in JACS, demonstrate how phosphorylated amino acids were incorporated into a designed β-hairpin peptide to study the effect on β-hairpin structure when the phosphate group is positioned to interact with a tryptophan residue on the neighboring strand. The three commonly phosphorylated residues in biological systems, serine, threonine, and tyrosine, were studied in the same β-hairpin system.
It was found that phosporylation destabilizes the hairpin structure by approximately 1.0 kcal/mol, regardless of the type of phosphorylated residue. In contrast, destabilization due to glutamic acid was about 0.3 kcal/mol. Double mutant cycles and pH studies are consistent with a repulsive interaction as the source of destabilization. These findings demonstrate a novel mechanism by which phosphorylation may influence protein structure and function.
Form Defines Function
Amanda Stewart, a PhD student in the Waters Group, has investigated the effects of β-hairpin structure on the binding affinity and selectivity for ssDNA versus dsDNA. Her research provides insights into the factors that contribute to the selective recognition of both ss- and dsDNA and suggests new approaches for designing biomimetic receptors.
The interactions involved in the binding of a designed β-hairpin dimer to single-stranded and duplex DNA were explored. Previously the peptide dimer had been found to bind ssDNA with a dissociation constant of 3µ through a combination of aromatic and electrostatic interactions, whereas binding to duplex DNA was primarily driven by electrostatic interactions. In her report, Amanda studied the effects of folding and chirality to determine the factors that contribute to affinity and selectivity for ssDNA versus dsDNA. Binding studies showed that 1) folding is crucial for binding to both ss- and dsDNA, and 2) chirality affects binding for duplex DNA but not for ssDNA. Taken together, these studies reveal different modes of binding for ss- and duplex DNA, with different driving forces, but in each case peptide structure contributes significantly to binding.
Cation-pi Models of the Histone Code
In work published in the Proceedings of the National Academy of Science, the Waters Group has demonstrated the critical role of cation-pi interactions in providing specificity to the interaction of Histone 3A with the HP1 Chromodomain.
Substitution of the trimethylamonium group of trimethyllysine in Histone 3A with a neutral tert-butyl analog abolishes binding despite the equivalent size and shape, indicating the importance of the cation-pi interaction over hydrophobicity. These results provide insight into the role of lysine methylation as part of the histone code for controlling gene expression.