Selective protein photocleavage by fluorescein derivatives.

Modification of the structure of small molecular probe which can act as photocleavage reagent has become a considerable challenge to improve the ability to target specific sites on a large protein. These photoreagents can provide valuable information on the binding site recognition and the mechanism of the photocleavage reaction under photochemical control. In this study, site specific photocleavage of lysozyme and avidin by fluorescein derivatives, fluorescein sodium salt (F-1) and 5(6)-carboxyfluorescein diacetate (F-2) were reported here for the first time. Functional groups on the photoreagent have been proven to effect on the interaction with the protein. Cleavage of the proteins by fluorescein derivatives were successful under visible region when irradiating the solution mixture of protein, fluorescein derivative and electron acceptor, cobalt (III) hexamine trichloride, at 490-492 nm. N-terminal amino acid sequencing of the cleaved fragments of lysozyme indicated the cleavage site between Trp108 - Val 109 for both probes, whereas the cleavage of avidin by F-1 and F-2 were detected between Trp70 - Lys71. Binding interaction can be investigated using methods as simple as absorption and fluorescence spectroscopies. Absorption and fluorescence studies indicated the strong binding interactions between fluorescein derivatives and the target proteins. Computational modeling was used to gain a better insight of the protein-probe binding interaction and binding sites. Molecular docking studies indicated that F-1 and F-2 were located near the hydrophilic and hydrophobic sites of both proteins within 4 Å away from the cleavage site. The docking results clarified the binding sites of F-1 and F-2 on proteins, corresponding to the results obtained from the protein photocleavage studies.

Tuning the chain length of new pyrene derivatives for site-selective photocleavage of avidin.

Rational design of photoreagents with systematic modifications of their structures can provide valuable information for a better understanding of the protein photocleavage mechanism by these reagents. Variation of the length of the linker connecting the photoactive moiety with the protein anchoring-group allowed us to investigate the control of the protein photocleavage site. A series of new photochemical reagents (PMA-1A, PMA-2A and PMA-3A) with increasing chain lengths is examined in the current study. Using avidin as a model system, we examined the interaction of these probes by UV-Vis, fluorescence spectroscopic methods, photocleavage and computational docking studies. Hypochromism of the absorption spectrum was observed for the binding of these new photochemical reagents with estimated binding constants (Kb) of 6.2 × 105, 6.7 × 105 and 4.6 × 105 M-1, respectively. No significant changes of Stern-Volmer quenching constant (Ksv) with Co(NH3)6Cl3 has been noted and the data indicated that the probes bind near the surface of the protein with sufficient exposure to the solvent. Photoexcitation of the probe-avidin complex, in the presence of Co(NH3)6Cl3, resulted in protein fragmentation, and the cleavage yield decreased with the increase in the linker length, and paralleled with the observed Ksv values. Amino acid sequencing of the photofragments indicated that avidin is cleaved between Thr77 and Val78, as a major cleavage site for all the three photoreagents. This site is proximate to the biotin binding site on avidin, and molecular docking studies indicated that the H-bonding interactions between the polar end-group of the photoreagents and hydrophilic amino acids of avidin were important in positioning the reagent on the protein. The major cleavage site, at residues 77-78, was within 5 Šof the pyrenyl moiety of the probe, and hence, molecular tuning of the linker provided a simple approach to position the photoreagent along the potential photocleavage site.

Photochemistry and mechanism of designed pyrenyl probe towards promoted cleavage of proteins.

A new photochemical reagent, succinic acid-1(1-pyrene)methylamide (PMA-SUC), was developed to recognize the specific binding sites on model proteins, egg-white lysozyme and avidin. The interaction of the photochemical reagent with the proteins was studied by UV-Vis, fluorescence spectroscopic methods and docking description. PMA-SUC was found to bind to lysozyme and avidin with binding constants (Kb) of 2.4×105 and 6.7×105 (M-1), respectively. The fluorescence intensity of PMA-SUC decreased with increasing concentration of both proteins. Quenching of PMA-SUC fluorescence, in the absence and presence of the protein by an electron acceptor (Hexaamminecobalt(III) chloride, Co(NH3)6Cl3) showed no significant changes in the Ksv values (Stern-Volmer quenching constant), indicating that PMA-SUC bound to the hydrophilic sites or near the surface of the proteins. Irradiation of protein-PMA-SUC mixture, at 342nm for a period of time, in the presence of Co(NH3)6Cl3 as an electron acceptor, resulted in the cleavage of both proteins with high specificity. Binding mechanisms were studied using Molecular docking method. Molecular docking study indicated the position of PMA-SUC upon binding to the proteins by hydrogen bonding interaction with donor-acceptor within the distance of less than 5Å in the minimum of binding free energy. The docking results have supported the results obtained from the spectroscopic methods and cleavage studies.

Use of a molybdenum(VI) complex as artificial protease in protein photocleavage.

In this study, a molybdenum(VI) peroxo α-amino acid complex, MoO(O2)2(α-leucine) (H2O), was prepared and used as an artificial protease for site-specific cleavage of porcine pepsin, a model protein. Cleavage of pepsin by MoO(O2)2(α-leucine) (H2O) was achieved under photochemical conditions at room temperature and pH 7.0. The reaction was activated by irradiation of the MoO(O2)2(α-leucine) (H2O)-protein mixture by UV light (320 and 340nm) for up to 30min. No cleavage was observed in the absence of MoO(O2)2(α-leucine) (H2O) or the light. The photocleavage yield increased with irradiation time. The cleaved fragments were sequencable, and the cleavage site was assigned to Leu(112)-Tyr(113). The cleavage reaction was quenched by ethanol. Therefore, hydroxyl radicals may be involved in the reaction and responsible for the cleavage of the protein. This is the first demonstration of the successful photocleavage of proteins by a molybdenum complex. This observation can provide a new approach for the photochemical footprinting of metal binding sites on proteins.

Selective cleavage of pepsin by molybdenum metallopeptidase.

In this study, the cleavage of protein by molybdenum cluster is reported for the first time. The protein target used is porcine pepsin. The data presented in this study show that pepsin is cleaved to at least three fragments with molecular weights of ∼23, ∼19 and ∼16 kDa when the mixture of the protein and ammonium heptamolybdate tetrahydrate ((NH(4))(6)Mo(7)O(24)·4H(2)O) was incubated at 37°C for 24h. No self cleavage of pepsin occurs at 37 °C, 24h indicating that the reaction is mediated by the metal ions. N-terminal sequencing of the peptide fragments indicated three cleavage sites of pepsin between Leu 112-Tyr 113, Leu 166-Leu 167 and Leu 178-Asn 179. The cleavage reaction occurs after incubation of the mixture of pepsin and (NH(4))(6)Mo(7)O(24)·4H(2)O) only for 2h. However, the specificity of the cleavage decreases when incubation time is longer than 48 h. The mechanism for cleavage of pepsin is expected to be hydrolytic chemistry of the amide bonds in the protein backbone.