Crambin is a small (46 amino acids) protein isolated from the seeds of the plant Crambe abyssinica. Crambin has been extensively used as a model protein for the development of advanced crystallography and NMR techniques and for computational folding studies. We set out to establish synthetic access to crambin. Initially, we synthesized the 46 amino acid polypeptide by native chemical ligation of two distinct sets of peptide segments (15 + 31 and 31 + 15 residues). The synthetic polypeptide chain folded in good yield to give native crambin containing three disulfide bonds. The chemically synthesized crambin was characterized by LC-MS and by 2D-NMR. However, the 31-residue peptide segments were difficult to purify, and this caused an overall low yield for the synthesis. To overcome this problem, we synthesized crambin by the native chemical ligation of three segments (15 + 16 + 15 residues). Total synthesis using the ligation of three segments gave more than a 10-fold increase in yield and a protein product of exceptionally high purity. This work demonstrates the efficacy of chemical protein synthesis by the native chemical ligation of three segments and establishes efficient synthetic access to the important model protein crambin for experimental studies of protein folding and stability.

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Flp provides a unique opportunity to apply the tools of chemical biology to phosphoryl transfer reactions. Flp and other tyrosine recombinases catalyze site-specific DNA rearrangements via a phosphotyrosine intermediate. Unlike most related enzymes, Flp's nucleophilic tyrosine derives from a different protomer than the remainder of its active site. Because the tyrosine can be supplied exogenously, nonnatural synthetic analogs can be used. Here we examine the catalytic role of Flp's conserved H305. DNA cleavage was studied using a peptide containing either tyrosine (pKa congruent with 10) or 3-fluoro-tyrosine (pKa congruent with 8.4). Religation was studied using DNA substrates with 3'-phospho-cresol (pKa congruent with 10) or 3'-para-nitro-phenol (pKa congruent with 7.1). In both cases, the tyrosine analog with the lower pKa specifically restored the activity of an H305 mutant. These results provide experimental evidence that this conserved histidine functions as a general acid/base catalyst in tyrosine recombinases.

Bacterial expression of full length β-amyloid (Aβ) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semi-synthetic method in which Aβ 1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Aβ 1-29 -Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Aβ 1-29 -MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Aβ 1-29 -MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH 2 -A30C-Aβ 30-40 , which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Aβ 1-29 -MESNA. Native chemical ligation proceeds rapidly and efficiently (> 90% yield) to give A30C-Aβ 1-40 . The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (> 90% yield) to give native sequence Aβ 1-40 . Overall, this system is highly efficient, and can yield ≈ 8-10 mg of pure Aβ 1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Aβ peptides. We have also expressed an Aβ construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Aβ 1-29 -Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15 N or 13 C labels for NMR. Future work will also include extending this system to longer Aβ peptides, such as Aβ 1-42 .

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