Steady-State and Time-Resolved Fluorescence Spectroscopic Studies on Interaction of the N-terminal Region with the Hairpin Loop of the Phytocystain Scb
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- Doi-Kawano, K., Nishimoto, E., Kouzuma, Y. et al. J Fluoresc (2009) 19: 631. doi:10.1007/s10895-008-0454-7
The steady-state and time-resolved fluorescece spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure.
KeywordsProteinase inhibitorCystatinTime-resolved fluorescenceFluorescence depolarizationSingle Trp protein
recombinant sunflower cystatin b
sunflower cystatin a
recombinant sunflower cystatin b of which Trp85 and Thr52 are replaced with Phe and Trp, respectively
recombinant sunflower cystatin b of which Trp85 and Thr58 are replaced with Phe and Trp, respectively
Cysteine protease inhibitors belonging to the cystatin superfamily have been found in several plant tissues, such as seeds, tubers, and ripening fruits; they are referred to as phytocystatin . Phytocystatins are single polypeptide chains with molecular weight from 10–12 kDa and share three conserved sequence motifs: Gly in the vicinity of the N-terminal region, Gln-Xaa-Val-Xaa-Gly in the first hairpin loop, and Trp in the second loop . Recently, the three-dimensional structure of oryzacystatin from rice seeds has been determinated by NMR, and showed that the structure is highly homologous to those of mammal cystatins .
The nature of the interaction of cystatin and protease has been extensively studied on mammal cystatins [3, 4]. The X-ray structure analysis of chicken cystatin showed that N-terminal segment and two hairpin loops are exposed on the surface of the protein, forming a wedge shaped edge . Docking model suggested that this wedge fits into the active site cleft of papain . This assumption was confirmed by structure analysis of cystatin B in complex with papain . In this structure, a wedge formed by the N-terminal end and two hairpin loops of cystatin B fits well into the active site cleft of papain. Although the functional implication of the first and second hairpin loops in the inhibitory activity was demonstrated by biochemical studies [7, 8], the contribution of the N-terminal region was controversial for some cystatins. Cystatin B derivative, in which 6 N-terminal amino acid residues including Gly were missing, was found to exhibit inhibitory activity, indicating that the N-terminal region is dispensable for inhibition . Pol and Björk described that mutation of Cys3 by Ser in human cystatin B caused reduced inhibitory activity, indicating the importance of the N-terminal region of the cystatin B . Furthermore, the N-terminal truncation of oryzacystatin had little effect on the inhibitory activity, suggesting that the N-terminal region is not involved in inhibitory activity . By contrast, subsequent mutagenesis study showed indispensability of the N-terminal region of oryzacystatin .
On the other hand, the implication of the N-terminal regions of cystatin A, cystatin C, and chicken cystatin in the inhibitory activity were well demonstrated by extensive biochemical studies [13–17]. It was recently described by kinetic study that cystatin A inhibits cysteine proteinases in two steps: the N-terminal region inhibits proteinases subsequent to other hairpin loops, and proposed that independent binding of the N-terminal region of cystatins to target proteinases after hairpin loops may be characteristic of most cystatin-proteinase interactions . In addition, Shibuya et al. found by NMR spectroscopic analysis of cystatin A that the mutation of the Gly4 residue caused chemical shifts of the Gln-Val-Val-Ala-Gly sequence, suggesting that the N-terminal region may be not only involved in direct interaction with proteinases, but also in maintaining the conformation of the first hairpin loop .
In our own study, two phytocystatins designated Sca and Scb were isolated from sunflower (Helianthus annuus) seeds. Sca and Scb consist of 80 and 98 amino acid residues, respectively, and exhibit inhibitory activity toward papain and fitin . Scb is a basic protein with the pI value of 10, and extra 5 amino acids in the middle of the molecule. The successive truncation of the N-terminal residues of Scb resulted in decreased inhibitory activity toward papain . These findings suggested that Scb inhibits proteinase in the manner similar to those of mammal cystatins. Fluorescence spectroscopy is an excellent method to study the correlation between the structure and function of protein. Especially, the time-resolved method with high sensitivity and time-resolution power enable to detect and analyze delicate but essential conformation and dynamics for the protein functions through the tryptophan residue of which location is specified in 3D structure of proteins. In this study, the interaction of the N-terminal region with the first and second hairpin loops was investigated using the steady-state and time-resolved fluorescence spectroscopic measurements of the recombinant Scb (rScb) and its mutant proteins. The results suggested that the N-terminal region plays an essential role in stabilizing the conformation of the first hairpin loop structure within Scb.
Materials and methods
Restriction enzymes and DNA modifying enzymes were purchased from MBI Fermentas and Toyobo, respectively. The oligonucleotide primers used in this study and thermo sequenase cycle sequencing kit containing 7-deaza-dGTP were obtained from GE Healthcare Bio-Science Corp. A chameleon double standard site-directed mutagenesis kit was from Strategene. Plasmid pGEM T-vector and expression plasmid pET-22b were obtained from Promega and Novagen, respectively. Escherichia coli strains JM109 and XlmutS were used as host cells for cloning and mutagenesis, and the strain BL21(DE3) was used as host cells for producing the recombinant Scb (rScb) as well as its mutant proteins. All other common chemicals and reagents were purchased at the highest purity available.
Mutagensis and DNA sequencing
For mutagenesis experiments, the cDNA coding for Scb in the plasmid pGEM T-vector was used. Site-directed mutagenesis was performed by the unique site elimination method developed by Deng and Nickoloff . All the mutations introduced were confirmed by DNA sequencing with a DNA sequencer (Shimadzu, DSQ-1000) using a thermo sequenase cycle sequencing kit (GE Healthcare Bio-Science Corp.).
Expression and purification of the recombinant proteins
The genes encoding wild-type and mutant Scb were expressed in E. coli strain BL21(DE3) using the expression plasmid pET-22b , as described in ref. 21. Proteins were purified from total extracts of the cells as previously described. The purity of wild-type and mutant enzymes was confirmed by reversed phase HPLC on a Cosmosil 5C4-300 (4.6 × 150 mm) and SDS-PAGE using 15% polyacrylamide gel. Deletions at the N-terminal residues were verified by N-terminal amino acid sequence using a gas-phase protein sequencer (Shimadzu, PSQ-1). Proteinase inhibitory assays were done, as previously described using the substrate Pyr-Phe-Leu p-nitrosoanilide . Concentration of rScb and its mutants were adjusted spectrophotometrically.
Steady state fluorescence
Every phytocystatin was prepared in 10 mM Na-phosphate buffer (pH 7.0). Steady-state fluorescence emission spectra were measured on Hitachi 850 fluorescence spectrophotometer. Each emission spectrum was strictly corrected for the excitation-detection response of the instrument and for the stray light.
Time-resolved fluorescence and fluorescence anisotropy studies
The fluorescence decay measurements were performed using the pico-second laser/multichannel plate based time-correlated single photon counting technique. The excitation source was a frequency-doubled output from cavity-dumped dye laser. The excitation pulse was a strain of 11 ps wide pulses wit a repetition rate of 800 kHz. The excitation wavelength was 300 nm, and the emission was detected after passing through a monochromator (H10, Jobin Yvon) with a 4 nm bandpass onto a multichannel plate photomultiplier tube (R1564, Hamamatsu). The channel width of the multichannel analyzer was 10 ps, and the data was collected in 2048 channels. The instrumentation used here was described elsewhere . Fluorescence decay data were analyzed by a nonlinear least squares iterative convolution method based on the Marquardt algorithm . Adequacy of the fitting was judged by inspection of the weighted residuals and of statistical parameters such as sigma and the serial variance ratio (SVR). For a satisfactory fit, the weighted residual must be randomly distributed and sigma and SVR should be in ranges of 1.0–1.2 and 1.8–2.0, respectively.
The fluorescence anisotropy decay measurement was performed by using the same instrument as fluorescence decay measurement. But, the parallel and perpendicular components were measured separately by orienting Glan-Taylor polarizers vertically and horizontally against the vertical excitation. The G-factor in the wavelength region 300–400 nm was determined by using azanaphthen in ethanol as an isotropic fluorescence standard. Adequacy of the fluorescence anisotropy decay was judged by the nonlinear least square curve fitting based on the Marquardt-Levenberg algorithm .
Results and discussion
Preparation and characterization of mutant proteins
Fluorescence spectroscopic studies using Trp as a fluorescence probe provide useful insight into the motion and interactions between the peptide elements of protein. Especially, specified the location of fluorophore in protein, the time-resolved study can provide the more quantitative information. Scb contains one Trp85 at the second loop structure. To probe the interaction of the N-terminal region with the first (positions 53–57: QVVAG) and second (positions 84–87: SWKH) loops by fluorescence spectroscopy, Trp was introduced around the first loop. For this purpose, Trp85 was first replaced with other amino acids or deleted, and the resulting mutants were characterized in term of the inhibitory activity.
Steady state and time-resolved fluorescence studies
When rScb, W85F/T52W, and W85F/T58W are excited at 300 nm, the fluorescence spectra due to W85, W52, and W58 are observed, respectively. They showed the fluorescence maxima at 352, 346, and 350 nm, respectively, to suggest that the tryptophan residues of three phytocystatins locate at the protein surface although W52 is in less polar circumstance (Fig. 3). The fluorescence of W85F/T52W showed much higher intensity than other two cystatins, rScb, and W85F/T58W. Only the fluorescence intensity of W85F/T52W was increased when the N-terminal deleting effects on the fluorescence spectra of the wild and mutant cystatin were examined. by the deleting of N-terminal amino acids.
Fluorescence decay parameters at 350 nm of cystatins a
The relative concentration of ground state Trp conformers of cystatins
Fluorescence anisotropy decay parameters of cystatins a
The motional freedom of tryptophan residue in each cystatin was shown in Table 3. By removing the N-terminal 4 amino acids, the freedom of the segmental motions of Trp85 and Trp58 in rScb and W85F/T58W increased a little, 5–10%. The motional freedom of rapid fluctuation of Trp52 in W85F/T52W-cystatin was smaller. But, it was increased more than 150% by removing the N-terminal 4 amino acids suggesting that the segmental motion of the peptide chain around Trp52 was much enhanced. The fastest correlation time dropped down to below 10 ps by the removing N-terminal 4 amino acids. These results reveal that the N-terminal amino acids do not only contribute to the packing structure in the local areas around Trp85 and Trp58 but also control the free fluctuation of the peptide chain by interacting with Trp52.
W85F/T52W and W85F/T58W exhibited the same inhibitory activities against the proteinase as rScb. Therefore, it is reasonable to consider that they reserved the essential structure for completing their function. At the same time, the elimination of the N-terminal amino acid residues similarly reduced their inhibitory activities. This fact demonstrates that the N-terminal residue plays an essential role for the inhibitory activity against the proteinase.
The time-resolved fluorescence studies on the rScb and other mutant phytocystatins used here consistently showed the N-terminal residue would approach and interact with Trp52. First of all, Trp52 in W85F/T52W showed very fast decay time in the order of 100 ps. This suggests that there exist the stronger fluorescence quenching side chain around Trp52. The strongest fluorescence quenching side chain is disulfide linkage which quenches the tryptophyl fluorescence through the electron exchange interaction. But its interaction is limited within the very short range. Other protonated amino group and deprotonated carboxylic group quench the tryptophyl fluorescence through the proton and electron transfer in the excited state, respectively. According to the anticipated X-ray crystallographic structure of Scb, there is no amino group and carboxylic group to quench the tryptophyl fluorescence around Thr52. Therefore, the N-terminal amino group must be approaching to Trp52 and one of the three conformers would strongly interact with N-terminal amino group. Because the DAS fraction corresponding to the fastest decay time was not recognized in the mutant of W85F/T52W of which N-terminal 4 amino acids were eliminated.