Total chemical synthesis of a functional interacting protein pair: The protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1

Generation of biological function by chemical methods is potentially of great importance for the understanding and targeting of physiological processes. Chemical synthesis of proteins offers the ability to alter the properties of target protein molecules in a tailor-made fashion. In the present work it is demonstrated that this methodology can be expanded to the elucidation of protein–protein interactions as exemplified by the complete chemical synthesis of the protooncogene product H-Ras as well as of the Ras-binding domain (RBD) of its effector c-Raf1. The 166-aa polypeptide chain of H-Ras was synthesized by native chemical ligation of three unprotected peptide segments. Similarly, the 81-aa RBD was prepared by ligation of two peptide segments. Both RBD and Ras displayed functional and spectroscopic properties indistinguishable from their recombinant forms as judged by CD spectroscopy and from transient kinetic measurements of the Ras–RBD interaction as well as from nucleotide replacement reactions in Ras. An unnatural amino acid bearing a nitrobenzofurazan side chain was introduced into position 91 of the RBD, providing unique fluorescence properties. The association transient of nitrobenzofurazan labeled with Ras⋅guanosine 5′-β,γ-imidotriphosphate showed a slow phase that had not been detected in earlier work by using other signals

Protein kinase casein kinase 2 holoenzyme produced ectopically in human cells can be exported to the external side of the cellular membrane

Ectokinases can phosphorylate extracellular proteins and external domains of membrane proteins influencing cell adhesion, movement, and cellular interactions. An ectokinase with the properties of casein kinase 2 (CK2) has been previously described, but little is known about the structural characteristics that allow this enzyme to be exported from the cell. Transfection of human embryonic kidney-293 cells with cDNAs coding for the catalytic (CK2α or CK2α′) and regulatory (CK2β) subunits with hemaglutinin tags allowed us to study the export of ectopically synthesized enzyme. When the catalytic (CK2α or CK2α′) and the CK2β regulatory subunits are cotransfected, the tetrameric enzyme composed of both subunits (holoenzyme) is detected outside the cell. This observation has been confirmed by assaying protein kinase activity in immunoprecipitates obtained with antihemaglutinin antibody by using a CK2-specific peptide substrate and by Western blots as well as by immunofluorescence of nonpermeabilized cells. Transfection with cDNA of catalytic or regulatory subunit alone does not result in export of these subunits. A study of the kinetics of appearance of the ectopically synthesized protein at different times after transfection indicates that a 5- to 7-h delay after the synthesis of the protein before it appears in the extracellular compartment. Using mutations of CK2α that eliminate phosphorylating activity [CK2α(Asp-156-Ala)] or that make it less sensitive to heparin inhibition [CK2α(Lys-75-Glu,Lys-76-Glu)] demonstrated that these mutations do not prevent the holoenzyme to be exported from the cells

Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet(7)]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the α-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by ≈45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments

Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein–intein junction

Protein splicing is a posttranslational autocatalytic process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. Although we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how these are catalyzed and regulated is less well developed. In the current study, a combination of NMR spectroscopy and segmental isotopic labeling has been used to study the structure of an active protein splicing precursor, corresponding to an N-extein fusion of the Mxe GyrA intein. The (1)J(NC′) coupling constant for the (-1) scissile peptide bond at the N-extein–intein junction was found to be ≈12 Hz, which indicates that this amide is highly polarized, perhaps because of nonplanarity. Additional mutagenesis and NMR studies indicate that conserved box B histidine residue is essential for catalysis of the first step of splicing and for maintaining the (-1) scissile bond in its unusual conformation. Overall, these studies support the “ground-state destabilization” model as part of the mechanism of catalysis