ROBERT RAMAGE - Bibliography from ROBERT RAMAGE. 4 October 1935 — 16 October 2019

Robert (Bob) Ramage was an innovative and versatile organic chemist who made pioneering contributions to the fields of target-oriented synthesis and synthetic methodology. His research programme was initially focused on the (bio)synthesis of natural products, but later expanded to include peptides and proteins, where he became a key figure in the development of that field in the UK and beyond. As a PhD student in Glasgow, he developed a synthesis of the sesquiterpene cuparene, an achievement that earned him a Fulbright scholarship to Harvard, where he worked under the guidance of R. B. Woodward ForMemRS. With Woodward he participated in the now classic synthesis of the beta-lactam antibiotic cephalosporin C. His independent career began in Liverpool, where he continued his interests in sesquiterpene synthesis, devising several notable syntheses. This period also saw him forge important collaborations with Alan Battersby (FRS 1966) and George Kenner FRS, with whom he worked on the biosynthesis of the alkaloid colchicine and the chemical synthesis of several polypeptides, respectively. In Manchester, and later in Edinburgh, he continued his work in peptide synthesis methodology, a multi-pronged effort that culminated in the successful chemical synthesis of crystalline ubiquitin, widely considered a landmark achievement in the field. His scientific impact reached beyond academia through various consultancies with the pharmaceutical industry and the founding of a successful biotech company. He also assumed various academic leadership roles throughout his career. Bob will be remembered for his exacting standards and resolve to tackle big problems, attributes that he instilled in the many young scientists he mentored. He is dearly missed by all who knew him, by his colleagues around the world and most of all by his family

Development of a Tandem Protein Trans-Splicing System Based on Native and Engineered Split Inteins

Protein trans-splicing involving naturally or artificially split inteins results in two polypeptides being linked together by a peptide bond. While this phenomenon has found a variety of applications in chemical biology and biotechnology, precious little is known about the molecular recognition events governing the initial fragment association step. In this study, fluorescence approaches have been used to measure the dissociation constant for the Ssp DnaE split intein interaction and to determine the on and off rates of fragment association. The DnaE fragments bind with low nanomolar affinity, and our data suggest that electrostatics make an important contribution to the very rapid association of the fragments at physiological pH. This information was used to develop a tandem trans-splicing system based on native and engineered split inteins. This novel system allows the one-pot assembly of three polypeptides under native conditions and can be performed in crude cell lysates. The technology should provide a convenient approach to the segmental isotopic or fluorogenic labeling of specific domains within the context of large multidomain proteins

Protein Semi-Synthesis in Living Cells

Incorporation of chemical probes into proteins is a powerful way to elucidate biological processes and to engineer novel function. Here we describe an approach that allows ligation of synthetic molecules to target proteins in an intracellular environment. A cellular protein is genetically tagged with one-half of a split intein. The complementary half is linked in vitro to the synthetic probe, and this fusion is delivered into cells using a transduction peptide. Association of the intein halves in the cytosol triggers protein trans-splicing, resulting in the ligation of the probe to the target protein through a peptide bond. This process is specific and applicable to cytosolic and integral membrane proteins. The technology should allow cellular proteins to be elaborated with a variety of abiotic probes

Biosynthesis of a Head-to-Tail Cyclized Protein with Improved Biological Activity

Biosynthesis of a Head-to-Tail Cyclized Protein with Improved Biological Activit

Protein Splicing Triggered by a Small Molecule

The use of small molecules that turn specific proteins on or off provides a level of temporal control that is difficult to achieve using standard genetic approaches. Consequently, the development of small-molecule switches of protein function is a very active area of chemical biology, sometimes referred to as chemical genetics. Most studies in this area rely on the identification of small molecules that bind directly to the active site of a target protein, thereby acting as agonists or antagonists of function. Strategies have also been described in which the small molecule triggers a change in the secondary, tertiary, or ternary structure of the protein, in so doing changing the functional state of the molecule. Another approach to this problem would be to alter the primary structure of a target protein in response to a small-molecule trigger; a dramatic change in primary sequence would be directly coupled to function. In principle, this can be achieved by harnessing protein splicing, a posttranslational editing process that results in the precise removal of an internal domain (termed an intein) from two flanking sequences termed the N- and C-exteins. In this communication we introduce a technique that allows protein splicing to occur only in the presence of the small molecule, rapamycin. This approach is expected to be independent of the nature of the two exteins and so should provide a general vehicle for controlling protein function using small molecules

A Chemical Probe for Protein Crotonylation

Protein lysine crotonylation has emerged as an important post-translational modification (PTM) in the regulation of gene transcription through epigenetic mechanisms. Here we introduce a chemical probe, based on a water-soluble phosphine warhead, which reacts with the crotonyl modification. We show that this reagent is complementary to antibody-based tools allowing detection of endogenous cellular proteins such as histones carrying the crotonylation PTM. The tool is also used to show that the histone acylation activity of the transcriptional coactivator, p300, can be activated by pre-existing lysine crotonylation through a positive feedback mechanism. This reagent provides a versatile and sensitive probe for the analysis of this PTM

Method for the Synthesis of Mono-ADP-ribose Conjugated Peptides

ADP-ribosylation is an important post-translational modification involved in processes including cellular replication, DNA repair, and cell death. Despite these roles, the functions of ADP-ribosylation, in particular mono-ADP-ribosylation, remain poorly understood. The development of a technique to generate large amounts of site-specific, ADP-ribosylated peptides would provide a useful tool for deconvoluting the biochemical roles of ADP-ribosylation. Here we demonstrate that synthetic histone H2B tail peptides, incorporating aminooxy or N-methyl aminooxy functionalized amino acids, can be site-specifically conjugated to ADP-ribose. These peptides are recognized as substrates by the ADP-ribosylation biochemical machinery (PARP1), can interact with the ADP-ribose binding proteins macroH2A1.1 and PARP9, and demonstrate superior enzymatic and chemical stability when compared to ester-linked ADP-ribose. In addition, the incorporation of benzophenone photo-cross-linkers into these peptides is demonstrated to provide a means to probe for and enrich ADP-ribose binding proteins

Simultaneous Triggering of Protein Activity and Fluorescence

Many areas of biology can benefit greatly from methods to spatially and temporally control protein activity. Here, we describe an approach that allows the simultaneous photo-triggering of the activity and the fluorescence of a protein. Smad2, a protein central to the transforming growth factor-β (TGF-β) signal transduction pathway, was modified with a fluorophore and a photocleavable moiety that acted as both a caging and a fluorescence quenching group. In its caged state, the protein formed a non-fluorescent heterodimer with the protein SARA. Irradiation with UV light and photocleavage of the caging group produced a fluorescent homotrimer. These in vitro experiments demonstrated that a photochemical trigger mimicking the critical biochemical event of serine phosphorylation involved in the TGF-β signaling pathway could be obtained and that fluorescence could be used as a read-out of protein activity. This approach should prove particularly useful for the monitoring of a protein's activity and location inside of living cells