ABSTRACT
A strategy inspired by tweezer receptors has been employed to develop a new host-guest system. The hybridization into a collagen-like triple helix is the driving force for the recognition that occurs with high affinity and selectivity. Several systems have been screened to find the best host-guest pair and this strategy may be implemented for tag fused protein recognition.
ABSTRACT
In order to understand the roles of protein matrix in electron transfer processes (ET) within biological systems, a heme-based donor (Zn-heme: ZnPP)-sensitizer (Ru2+(bpy)3)-acceptor (cyclic viologen: BXV4+) triad 1 was used as a probe molecule. Two semi-synthetic systems, Cyt-b562(1) and Mb(1), in which the triad is incorporated into cytochrome b562 (Cyt-b562) or into myoglobin (Mb), were constructed by cofactor reconstitution. These two semi-synthetic proteins were compared with the triad itself (i.e., without the protein matrix) using absorption spectroscopy, steady state emission and excitation studies, laser flash photolysis experiments, and molecular modeling. Photoexcitation of the ZnPP moiety of Cyt-b562(1) or Mb(1) leads to a direct ET from the triplet state of ZnPP state (3ZnPP) to BXV4+ to generate a final charge-separated (CS) state, Cyt-b562(Zn+)-Ru2+-BXV3+* or Mb(Zn+)-Ru2+-BXV3+*. On the other hand, direct ET from the excited ZnPP moiety to the BXV4+ moiety is also involved in 1 in the absence of the protein matrix, but the excited state of ZnPP involved is not 3ZnPP, but the singlet excited state (1ZnPP) in this pathway. When the Ru2+(bpy)3 moiety of Cyt-b562(1) or Mb(1) is excited, a stepwise ET relay occurs with the ion-pair, Cyt-b562(Zn)-Ru3+-BXV3+* or Mb(Zn)-Ru3*-BXV3+*, as an intermediate, leading to the same final CS state as that generated in the direct ET pathway. The lifetimes of the corresponding final CS states were determined to be 300 ns for 1 in the absence of the protein matrix, 600-900 ns for Cyt-b562(1) and 1.1-18 micros for Mb(1), the values of which are greatly affected by the protein matrix. Molecular modeling study of the three systems consistently explained the differences of their photophysical behavior.
Subject(s)
Cytochrome b Group/chemistry , Escherichia coli Proteins , Myoglobin/chemistry , Electrons , Kinetics , Lasers , Models, Molecular , PhotolysisABSTRACT
Recent developments in new bioorganic methodologies have greatly facilitated the site-specific incorporation of non-natural amino acids into the protein framework. It is now desirable for chemists to explore promising concepts based on chemistry for regulation and extension of functions of naturally occurring enzymes using non-natural molecules, in order to promote the new trends in protein/enzyme engineering. This article demonstrates that the concepts of host-guest (or supramolecular) chemistry, which have been developed over the last few decades, provide powerful tools for the artificial control of the functions of native proteins and enzymes.
Subject(s)
Enzyme Stability , Protein Conformation , Protein Engineering , Proteins/chemistry , Ribonucleases/chemistry , Amino Acid Sequence , Binding Sites , Kinetics , Ligands , Light , Metals/chemistry , Models, Molecular , Molecular Sequence Data , Molecular Structure , Myoglobin/chemistry , Myoglobin/metabolism , Protein Binding , Proteins/chemical synthesis , Ribonucleases/genetics , Ribonucleases/metabolismABSTRACT
Spermine-sensitive stabilization of semisynthetic Ribonuclease S' was successfully carried out by sequence specific incorporation of a poly-anion domain into alpha-helix region of S-peptide.
Subject(s)
Ribonucleases/chemistry , Ribonucleases/chemical synthesis , Spermine/chemistry , Amino Acid Sequence , Kinetics , Models, Chemical , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Denaturation , Protein Structure, Secondary , TemperatureABSTRACT
The sugar-facilitated structure and enzymatic activity change of engineered myoglobins bearing a phenylboronic acid moiety, which were semisynthesized by a cofactor reconstitution method, were studied by the denaturation experiment, spectrophotometric titration of the pKa shift of the axial H2O, circular dichloism (CD), and the kinetics of the myoglobin-catalyzed-aniline hydroxylation reaction. Both boronophenylalanine-appended myoglobin [Mb(m-Bphe)2] and phenylboronic acid-appended myoglobin [Mb(PhBOH)2] were stabilized by approximately 2 kcal/mol upon complexation with D-fructose. CD spectral changes and the sugar-induced pKa shift suggested that the microenvironment of the active site of these myoglobins was re-formed from a partially disturbed state to that comparable to the native state upon D-fructose binding. The correlation of pKa with kcat (for the aniline hydroxylase activity) and the delta GDH2O-kcat profile showed that these structural changes of Mb-(m-Bphe)2 and Mb(PhBOH)2 were closely related to their sugar-enhanced aniline hydroxylase activity. Thus, the results established that an incorporation of the artificial receptor molecule can be a valid methodology for the design of stimuli-responsive semiartificial enzymes.
