Construction And Discriminating Application Of Pyrene-based Fluorometric Probe-pair For Proteins

To make sensitive and specific biomolecular sensors is crucial for characterization of biomolecular interactions. It is also important for disease diagnostics, environmental monitoring and quality control in food production. Recently, fluorescence probes for proteins have been widely used to quickly detect the existence of protein and/or dynamic aspects of protein structure. The use of fluorescent probes has apparent advantages over other methods in sensitivity, selectivity and performance. In addition, fluorescent probes can be imaged in vivo, even in single living cells.Melittin is a cationic hemolytic peptide composed of 26 amino acids (NH2-GIGAVLKVLTTGLPALISWIKRKRQQ-CONH2). It is the main toxic component in the venom of the European honey bee. Melittin is water soluble and has the ability to spontaneously associate with natural and artificial membranes because of the amphiphilic property. So it is widely used as a convenient model for monitoring lipid-protein interactions in membranes, and also employed as a paradigm for protein-lipid interaction.We designed and synthesized N-[4(1-pyrene)-butyroyl]-L-tryptophan (PLT; 1) for recognizing melittin by combining a pyrene-containing fluorophore, 4-(1-pyrenyl)-butyric acid, with a tryptophan (recognizing group). 1 exhibited high sensitivity and selectivity for melittin in aqueous solution with the responded signal of specific excimer of pyrene and very low detected limit (0.5μM). This can be used in direct dectecting melittin in aqueous.Initial experiment was performed to detect whether an interaction of 1 and melittin occurred, which was proved by the changes in the emission max both for Trp in melittin and pyrene in 1. The interaction of 1 with melittin was also determined by measuring the fluorescence emission of 1 when bound to melittin. When excited by 340 nm, 1 alone had strong emission bands at 376 and 396 nm attributing to pyrene monomer. Further, the addition of 0.1 equivalent melittin to the solution containing 1 showed a new peak around 475 nm that obviously increased with the gradual addition of melittin. Meanwhile, a gradual decrease of emission intensity in the region of 370-400 nm was also clearly observed. We use the value of the fluorescence intensity ratio I475/I376 as a function of the concentration of melittin to demonstrate the responsive ability of 1 to melittin. 0.5μM melittin can be detected easily with a value of I475/I376 close to 0.1. When the concentration of melittin was increased up to 2.5μM, the value of I475/I376 became constant, suggesting a saturated binding between 1 and melittin where 4 mol of 1 may be bound per mol of melittin. Such a proposed ratio was further proved by the method of continuous variation (Job's plot).It is proposed that an electrostatic interaction can be formed between melittin and 1 as the involved basic sequence in melittin (K and R) in former and carboxylic group in later, and therefore induces the formation of ground-state dimmer (GSD) of pyrene at its surface. If such a proposal is correct, similar phenomenon should be occur at the surface of other proteins with high pI as melittin (for example, lysozyme, ribonuclease A and cytochrome C) and do not occur for the proteins with middle pI (as hemoglobin, myoglobin) or low pI (BSA, insulin) at identical neutral condition. To test the recognize mechanism, we use other proteins such as BSA, ribonuclease A, lysozyme,hemoglobin and Cytochrome C (sorted by pI) to test the interaction with 1. But all these proteins can not induce 1 to form ground-state dimer (GSD). Then, we adapt the conformation of melittin by adjusting the salt or pH. We found the change in secondary structure have no effect in recognizing. We also designed and synthesized the analog of 1, N-[4(1-pyrene) butyroyl]- L -phenylalanine (PLP; 2) and N-[4(1-pyrene)-butyroyl]-L-glutamic acid (PLG; 3), but after the displacement of the tryptophan, melittin can not induce 2 or 3 to form GSD, this means that the indole ring of tryptophan is very important in recognizing.In order to get the peptide sequences which can interact with 1, we designed several peptides, such as N-terminal of melittin (from 1 to 14 aa), C-terminal of melittin (14-26 aa), the positive charge region of melittin (20-26 aa) and so on. Data shows that only the charge interaction can not induce 1 to form GSD. The Trp19 in melittin showed very important role to induce 1 to GSD. The mutant C-terminal 13 aa peptide of melittin in Trp19 to Ala19 can not induce 1 to GSD. The binding of adjacent -KRKR- region in melittin to 1 may facilitate an ideal opportunity for pyrene GSD formation between four 1 at melittin surface. A careful investigation of 1 binding to peptides of melittin indicated two structural requirements for these changes in fluorescence. First, an ionic interaction of the anion in 1 and Arg/Lys residues (close to C-terminal). Second, a complex of the peptide and 1 provides the necessary"structured"environment for the formation of pyrene GSD.We measured CD spectra to test if the addition of 1 to melittin can induce a conformation change of melittin or other peptides. Observed from the spectra, at the low concentration of such peptides, it showed that the addition of 1 to these peptides does not change the secondary structure.Then we designed, expressed and purified the GST-Melittin-C-13 by E.coli expression system and found the expressed protein can induce 1 to GSD. The results suggested that Mlt-C-13 can be used as a tag for target protein detection, with the further assistence of 1. Such a study using 1 as a probe model provided a foundation for further biophysical examination of recognition and mechanisms clarify between it and proteins.We also designed some analogs such as N-terminal and C-terminal of HPV16-L1 and HPV16-L2 to test if these peptides can interact with 1. Data shows that only the HPV-16 L2CtW can induce 1 to form GSD, which means 1 may be used as a fluorescence probe to directly detect HPV.In conclusion, we use a single amino acid linked with pyrene as a biosensor for recognition of melittin by monitoring the pyrene GSD fluorescence peak. Sub-micromolar level (< 0.5μM) of melittin can be detected easily at neutral pH in aqueous. Furthermore, the specific region of PALISWIRKRKQQ in the C-terminal of melittin induced the formation of pyrene GSD efficient, such a region can be used as a labeling peptide to target protein and further detection by using 1.