Julio A. Camarero
Rockefeller University
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Featured researches published by Julio A. Camarero.
Angewandte Chemie | 1998
Julio A. Camarero; Joanna Pavel; Tom W. Muir
Extremely fast cyclization of the linear polypeptide precursor 1 takes place to form 2. The reaction appears to be assisted by the native fold of 1, which positions the reactive ends in close proximity. The circular topology has no influence on the folding or function of 2.
Biopolymers | 1999
Brenda Ayers; Ulrich K. Blaschke; Julio A. Camarero; Graham J. Cotton; Mandë Holford; Tom W. Muir
Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/rhodamine-containing construct (12) and the ketone-containing construct (14). The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.
Bioorganic & Medicinal Chemistry | 2001
Julio A. Camarero; David Fushman; David Cowburn; Tom W. Muir
Here we describe the first example of a peptide chemical ligation reaction performed inside a living cell. A cell-based native chemical ligation approach was developed and used to generate a circular version of the N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein inside Escherichia coli cells. The in vivo cyclization reaction was extremely efficient and the resulting circular protein domain was fully biologically active and able to adopt the native SH3 folded structure. This work represents an important step towards the in vivo generation of small backbone cyclic peptides for use in basic biological research.
Chemical Communications | 1997
Julio A. Camarero; Tom W. Muir
A fully unprotected 15 residue peptide containing an N-terminal Cys residue and a C-terminal thioester moiety is cleanly converted to the corresponding head-to-tail cyclic peptide in aqueous buffers at around neutral pH.
Letters in Peptide Science | 2000
Julio A. Camarero; Alberto Adeva; Tom W. Muir
This paper describes a practical new use of 3-mercaptopropionic acid as a highly versatile multidetachable linker for solid-phase synthesis. Our approach is based on the stability of the alkylthioester functionality to optimized Boc-SPPS protocols and HF treatment, as well as on the mild activation of the thioester functionality toward nucleophilic or reductive displacement. This allows several C-terminal modifications to be introduced into a synthetic molecule during the cleavage step. We have shown that unprotected peptides can be efficiently cleaved from a propyl thioester-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer resin using a great variety of nucleophiles to give the corresponding C-terminally modified peptides (esters, thioesters, carboxylic acids, thioacids, amides, hydroxamic acids, hydrazides, alcohols). The nucleophilic cleavage reaction is both rapid and exceptionally clean in all the cases tested.
Current protocols in protein science | 2001
Julio A. Camarero; Tom W. Muir
The total synthesis and semisynthesis of proteins allows the site‐specific incorporation of unnatural amino acids, post‐translational modifications, and biophysical/biochemical probes into the target molecule. Among the various chemical and enzymatic approaches available for the synthesis/semisynthesis of proteins, the native chemical ligation technique has proven especially useful and is the exclusive focus of this unit. This unit first discusses how to choose the ligation site(s) in the target protein and then outlines how to obtain the necessary polypeptide building blocks using either chemical synthesis or recombinant DNA expression. Next, the synthesis of a protein by native chemical ligation of two polypeptide fragments is described. The synthesis of a protein from three polypeptide fragments using a sequential native chemical ligation strategy is also described. Support protocols describe how to obtain the necessary polypeptide fragments using either chemical synthesis or recombinant DNA expression.
Biochemistry | 1998
Julio A. Camarero; Brenda Ayers; Tom W. Muir
A novel technique is described that allows the synthesis, functional analysis, and quantitative readout of defined arrays of polypeptide analogues in aqueous solution. Key to this approach is the use of a simple encoding-decoding system in which a unique Fmoc-amino acid tag is covalently attached to the C terminus of each member of a molecular array through a selectively cleavable bond. These tags can be cleanly removed from the molecules they encode, allowing single-step characterization and quantification of the entire mixture by HPLC. The utility of this technique is illustrated through the preparation of an array of proline-rich sequences based on the exchange factor C3G, one of the natural ligands of the N-terminal SH3 domain from the proto-oncogene, c-Crk. The array was designed to systematically modify those residues within the C3G peptide ligand thought to make key interactions with the c-Crk SH3 domain. Using competition binding experiments, it was possible to determine the relative ED50 values for the entire array of molecules simultaneously. These studies revealed that in order to maintain optimal binding to the SH3 domain, the P-3 side chain of the ligand must be positively charged and the P-0 side chain must be hydrophobic and extend beyond the gamma-carbon. The excellent correlation between these relative ED50 values and a series of relative Kd values determined from individual peptides suggests that this approach may be useful in determining, in a parallel fashion, the relative biological activities of arrays of polypeptides.
Journal of Peptide Research | 2009
Julio A. Camarero; Graham J. Cotton; Alberto Adeva; Tom W. Muir
Journal of Molecular Biology | 2001
Julio A. Camarero; David Fushman; Satoshi Sato; Izabela Giriat; David Cowburn; Daniel P. Raleigh; Tom W. Muir
Journal of Molecular Biology | 2001
Leonid Minakhin; Julio A. Camarero; Mandë Holford; Christian N. Parker; Tom W. Muir; Konstantin Severinov