Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency

Normalized CD spectra of Tat1 analogs in which the pharmacophore residues were substituted by either Ala or Tic-Oic moiety.

<p>Normalized CD spectra of Tat1 analogs in which the pharmacophore residues were substituted by either Ala or Tic-Oic moiety.</p

Chemical shift variation plot between Hα resonances of major conformations of Tat1, Tat1_4-5TO, Tat1_8-9TO, Tat1_8-9TOD, Tat1_4,5TO,8-9TOD and Tat1_A4-5,8–9.

<p>Chemical shift variation plot between Hα resonances of major conformations of Tat1, Tat1_4-5TO, Tat1_8-9TO, Tat1_8-9TOD, Tat1_4,5TO,8-9TOD and Tat1_A4-5,8–9.</p

The second derivative of FTIR spectra of Tat1, recorded in H₂O, D₂O and in a film mode.

<p>The second derivative of FTIR spectra of Tat1, recorded in H₂O, D₂O and in a film mode.</p

Overlaid fragments of TOCSY (green-blue) and ROESY (red-yellow) spectra of Tat1_8-9TO (A) and Tat1_8-9TOD (B).

<p>Overlaid fragments of TOCSY (green-blue) and ROESY (red-yellow) spectra of Tat1_8-9TO (A) and Tat1_8-9TOD (B).</p

The Ala-scan analogs of Tat1 diversely affect the ChT-L peptidase of the 20S proteasome.

<p>The Ala-scan analogs of Tat1 diversely affect the ChT-L peptidase of the 20S proteasome.</p

Superposed conformations obtained in the last 800 ps of MD simulations and surface electrostatic potential maps.

<p>The maps indicate regions of negative potential in red, positive potential in blue, and neutral potential in white. The figures were generated by the MOLMOL). (<b>A</b>) Tat1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143038#pone.0143038.ref036" target="_blank">36</a>], (<b>B</b>) Tat1_4-5TO, (<b>C</b>) Tat1_8-9TO, (<b>D</b>) Tat1_8-9TOD, (<b>E</b>) Tat1_4-5TO,8-9TOD and (<b>F</b>) Tat1_A4-5,8–9. RMSD values are 0.800, 1.008, 1.357, 1.187, 0.865 and 1.140 Å for the backbone atoms in the fragment 1–9 of Tat1, Tat1_4-5TO, Tat1_8-9TO, Tat1_8-9TOD, Tat1_4-5TO,8-9TOD and Tat1_A4-5,8–9, respectively.</p