The influence of coiled-coil motif of serine recombinase toward the directionality regulation

Serine integrases promote the recombination of two complementary DNA sequences, attP and attB, to create hybrid sequences, attL and attR. The reaction is unidirectional in the absence of an accessory protein called recombination directionality factor. We utilized tethered particle motion (TPM) experiments to investigate the reaction behaviors of two model serine integrases from Listeria innocua phage LI and Streptomyces coelicolor phage C31. Detailed kinetic analyses of wild-type and mutant proteins were carried out to verify the mechanisms of recombination directionality. In particular, we assessed the influence of a coiled-coil motif (CC) that is conserved in the C-terminal domain of serine integrases and is an important prerequisite for efficient recombination. Compared to wild type, we found that CC deletions in both serine integrases reduced the overall abundance of integrase (Int) att-site complexes and favored the formation of nonproductive complexes over recombination-competent complexes. Furthermore, the rate at which CC mutants formed productive synaptic complexes and disassembled aberrant nonproductive complexes was significantly reduced. It is notable that while the φC31 Int CC is essential for recombination, the LI Int CC plays an auxiliary role for recombination to stabilize protein-protein interactions and to control the directionality of the reaction

ESR study of interfacial hydration layers of polypeptides in water-filled nanochannels and in vitrified bulk solvents.

There is considerable evidence for the essential role of surface water in protein function and structure. However, it is unclear to what extent the hydration water and protein are coupled and interact with each other. Here, we show by ESR experiments (cw, DEER, ESEEM, and ESE techniques) with spin-labeling and nanoconfinement techniques that the vitrified hydration layers can be evidently recognized in the ESR spectra, providing nanoscale understanding for the biological interfacial water. Two peptides of different secondary structures and lengths are studied in vitrified bulk solvents and in water-filled nanochannels of different pore diameter (6.1~7.6 nm). The existence of surface hydration and bulk shells are demonstrated. Water in the immediate vicinity of the nitroxide label (within the van der Waals contacts, ~0.35 nm) at the water-peptide interface is verified to be non-crystalline at 50 K, and the water accessibility changes little with the nanochannel dimension. Nevertheless, this water accessibility for the nanochannel cases is only half the value for the bulk solvent, even though the peptide structures remain largely the same as those immersed in the bulk solvents. On the other hand, the hydration density in the range of ~2 nm from the nitroxide spin increases substantially with decreasing pore size, as the density for the largest pore size (7.6 nm) is comparable to that for the bulk solvent. The results demonstrate that while the peptides are confined but structurally unaltered in the nanochannels, their surrounding water exhibits density heterogeneity along the peptide surface normal. The causes and implications, especially those involving the interactions between the first hydration water and peptides, of these observations are discussed. Spin-label ESR techniques are proven useful for studying the structure and influences of interfacial hydration

Long-range water accessibility study by ESE.

<p>The theoretical fits (red lines) to the ESE experimental data (blue lines) using a stretched exponential function (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>) as previously described. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone.0068264-Huang1" target="_blank">[11]</a> The results for the n3β-s and PPm3-s are shown in (a) and (b), respectively. The decay signals acquired by the ESE experiments were fitted over the maxima of the deuterium modulation as described in Zecevic et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone.0068264-Zecevic1" target="_blank">[32]</a> to minimize the influence from destructive interference of nuclear modulations. The obtained values of the T_M (in ns) and stretching exponent x are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>. The T_M values can be directly used to yield the surrounding proton density (<i>C_ex</i>; cf. Eq. 2) within the range of ∼2 nm from a nitroxide spin.</p

Water accessibility study of the PPm3-s by ESEEM.

<p>(a) Three-pulse ESEEM time-domain data (solid lines) after the removal of the exponential decaying function in the raw data. The modulation depth is directly correlated to the peak intensity of the FT-ESEEM and can be quantitatively characterized by the best-fit parameter <i>k_D</i> (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-t001" target="_blank">Table 1</a>). The dashed lines represent the theoretical fits to the experimental data using the equation described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-g003" target="_blank">Figure 3</a>. (b) The FT-ESEEM data for the PPm3-s in various deuterated conditions. The peaks correspond to the Larmor frequency of nucleus ²H, indicating the PPm3-s is surrounded by D₂O. The inset shows a ribbon model of a PPm3 variant carrying three spin labels.</p

Parameters obtained in the analyses of the ESE and ESEEM data.^§

§<p>Estimated errors: 5%(T_M), 10%(x), 13% (<i>C_ex</i>), 5% (<i>k_D</i>), 10% (Π). Abbreviations: <b>n3-s-a</b> (the n3-s is within SBA15a containing pure water); <b>n3-s-b</b> (the n3-s is within SBA15b containing pure water); <b>n3-s-sol(s)</b> (the n3-s is in a vitrified bulk solvent containing 40 wt% sucrose, (s), in D₂O or H₂O); <b>PPm3-s-sol(g)</b> (PPm3-s is in a vitrified bulk solvent containing 40v/v% glycerol in H₂O; deuterated glycerol is used if the solvent is D₂O, a condition of which is represented by sol(dg)/D₂O in main text); <b>PPm3-s-sol(s)</b> (PPm3-s is in a vitrified bulk solvent containing 40 wt% sucrose in D₂O or H₂O). In all of the experiments, the surface group of the nanochannels is modified to –SiOD in advance if D₂O is used. See Method for details.</p>#<p>The values of T_M and x are obtained in the analysis of the pulsed ESE measurements using a stretched exponential function, , where τ is the time between the two pulses, x the exponent, and Y(0) is the echo intensity at τ  = 0. The obtained values are used to yield <i>C_ex</i> using Eq. (2). The <i>C_ex</i> represents ESE-based water accessibility within the range of ∼2 nm from the nitroxide spin.</p>¶<p>The <i>k_D</i> values are obtained in the theoretical analysis of the ESEEM measurements as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068264#pone-0068264-g003" target="_blank">Figure 3</a>. The best-fit values for the damping constant (<i>τ₀</i>) and phase (φ) are very close together (2.9∼3.0). The Π represents ESEEM-based water accessibility within the range of ∼0.35 nm from the nitroxide spin.</p

Determination of the n3α-d structure.

<p>(a) The time-domain DEER signals of the studied conditions. The gray lines represent the exponential baselines that best fit the data. Inset shows a ribbon model of the n3α-d derived from NMR data (PDB code: 1M25). (b) The P(r) distributions extracted from the time-domain DEER data by the Tikhonov regularization analysis. The average distances (∼2.0 nm) are consistent with the expectation. (c) The cw-ESR spectra of the n3α-d. The spectra of the bulk solution studies are characterized by a broader linewidth and the spectral heterogeneity (indicated by arrows) as compared to the spectra of the nanochannel studies.</p

Determination of the n3β-d structure.

<p>(a) The time-domain DEER data for the n3β-d (0.5 mM) in the studied conditions, including the vitrified bulk solvent (sol(s)/H₂O) and the nanochannels (SBA15a and SBA15b). The gray lines represent the exponential baselines that best fit the DEER data. There are two insets. One displays a ribbon model for the n3β-d showing the spin-label side-chains at the 3rd and 9th sites of the peptide. The model was derived from a NMR study (PDB code: 1G04). The other inset shows the baseline-corrected DEER traces for the sol(s)/H₂O and the SBA15a, and also the simulated DEER traces (in green color) using the obtained P(r)s. There are some distinct differences in the two traces. (b) The (normalized) interspin distance distributions of the n3β-d peptides in the conditions studied. The average distances of the three measurements are approximately the same, indicating the n3β structure remains roughly unchanged. A much-broadened P(r) for the bulk solution study is obtained due to the solvent heterogeneity. The inset shows the Pake doublets converted from the DEER data. (c) Cw-ESR spectra of the n3β-d at 50 K. The clustering, caused by the solvent heterogeneity at 50 K, is evidently observed in the cw-ESR spectra of the bulk solution study, but not in the nanochannel studies.</p