In the present work, we have reported for the first time the biochemical and biophysical characterization of a putative SSB from bacteriophage Phi11, annotated as rGp13. Here, we have demonstrated a detailed study on the effect of various parameters (pH, ions, length of the oligonucleotide, etc.) on the structure and function of Phi11 rGp13 by employing far UV-CD, fluorescence spectroscopy and EMSA.
We studied the influence of varying pH on the secondary structure and function of rGp13 in the range of pH 4–10. Far UV-CD data revealed that the protein is stable between pH 5–10. However, at pH 4, the spectrum showed destabilisation in the rGp13 structure, possibly resulting from a decrease in the Antiparallel β-sheet component and a significant increase in the parallel β-sheet component. The results are concomitant with the EMSA data, where rGp13 showed efficient binding with ssDNA between the pH 5–10 range but showed no binding at pH 4 (Fig. 1A-C). The reason for this stability possibly lies in the Antiparallel β-sheet component of the rGp13, which exhibits a very slight change in the presence of buffers with different pH (pH 5–10). Antiparallel β-sheets are the major component of the SSB proteins, which form the OB-fold. The OB-fold is normally comprised of two, three, or five-stranded antiparallel β-sheets and is often described as a Greek key motif [11, 27]. The β-sheets are arranged to form a β-barrel that is typically capped by an α-helix at one end and a binding cleft at the other [12]. Thus, changes in the composition of the β-sheets might change the overall behaviour of the protein.
The data obtained from the studies done in the presence of monovalent cations showed that Na+, K+, Li+ and NH4+ had a stabilising effect on the structure of rGp13. The nucleic acid binding assay also showed that rGp13 has an almost similar binding affinity for ssDNA in the presence of all the monovalent cations used, with K+ and NH4+ being the better options ( Fig. 2A-C, Supplementary Figure S2). This data suggests that rGp13 is functionally active in the presence of Na+, K+, Li+ and NH4+ ions.
Among the divalent cations, rGp13 is completely destabilised in the presence of Zn2+ ions with a complete loss of the α-helical and parallel β-sheet content. Ni2+ ions only slightly destabilised the protein, as evident from the CD spectra data. The CD spectra data further indicate that rGp13 is structurally stable in the presence of Mn2+, Mg2+, and Ca2+ ions. However, further analysis of the secondary structure content of the protein unveiled a decrease in the α-helical content in the presence of Mn2+ ions. We performed a nucleic acid binding assay with the divalent cations to establish a structure-function relationship. Among the studied divalent cations, Zn2+ was found to severely hamper the rGp13-ssDNA interaction as was expected from its CD spectra. This finding indicates that not only antiparallel β-sheet but also a reduction in the α-helical content too can hinder the rGp13-ssDNA interaction. At neutral pH, Zn2+ ions can degrade nucleic acid, as shown by Butzow et al. [28]. EMSA even showed a partial degradation of nucleic acid induced by Zn2+ ions. A super shift was observed in the case of Ni2+ ions, suggesting there might be a formation of higher oligomers. The divalent cations Mn2+, Mg2+, and Ca2+ did not affect the rGp13-ssDNA interaction, and the best binding was observed in the presence of Mg2+ ions (Fig. 3A-C, Supplementary Figure S3). It is very clear from Table 3 that the presence of Mn2+ ions led to a complete loss in parallel β-sheet and a partial loss in the α-helical content, but it did not affect the binding of rGp13 with the ssDNA. Thus, maintaining a specific ratio of α-helix and Antiparallel β-sheet is crucial for both efficient rGp13-ssDNA interaction and overall structural stability. The α-helical content seems to play a role in maintaining the structural stability of the rGp13 as well as in DNA binding. As mentioned earlier, an OB-fold commonly comprises an α-helical cap [12]. More interestingly, our research revealed that a partial loss in the α-helical content did not have any effect on the rGp13-ssDNA interaction, as observed in the case of Mn2+ ions. Our study also suggested that changes in parallel β-sheet content had little or no impact on rGp13 activity (Tables 1, 2 and 3).
To validate our Far UV-CD data, we further examined the influence of changes in buffer conditions (pH and ions) on the tertiary structure of rGp13 using fluorescence studies. rGp13 has been found to maintain its tertiary structure over a wide range of pH ranging from pH 5–10. However, a slight increase in the fluorescence intensity and a shift in the emission maxima was observed at pH 10. The acidic pH 4 has resulted in a complete destabilization of the tertiary structure, as evident from the shift in the emission spectrum (Fig. 1C). Among the monovalent and divalent cations only Zn2+ ions resulted in a significant shift in the emission maxima as well as a decrease in the fluorescence intensity. The results obtained from the fluorescence spectroscopy corroborated with CD and EMSA data. It is evident that rGp13 is an exceptionally robust protein, which undoubtedly makes it perfect for a wide range of biotechnological applications.
To further investigate the ssDNA binding properties of rGp13, we conducted an EMSA using single-stranded dT-oligonucleotides of various lengths. No specific binding was observed with (dT)15 and (dT)20 (Fig. 4A and B). However, longer single-stranded oligo-(dT), such as (dT)25 and (dT)30 (Fig. 4C and D), yielded a single band with reduced mobility, even at higher protein concentrations. TaqSSB, TthSSB and DmuSSB form two or more complexes when incubated with (dT)70 and (dT)76 with increasing protein concentrations. [29, 30]. NeqSSB formed at least five ssDNA-protein complexes even with oligonucleotides as small as (dT)35 [31]. However, rGp13 exhibited a distinct behaviour compared to other SSBs. Even when a saturating concentration of rGp13 was incubated with (dT)50 and (dT)100 (Fig. 4E and F)., it showed a single shifted band. This behaviour was independent of the length of the oligonucleotide, salt concentrations and buffer conditions, suggesting a non-cooperative binding mode (Fig. 5). We have also shown that rGp13 can bind with dsDNA as well. It is worth noting that rGp13 exhibited a high degree of affinity towards dsDNA, similar to that of the ssDNA (Fig. 6). This observation suggests that rGp13 could potentially serve as a valuable tool in further DNA-related research. Also, SSB binding to multiple substrates likely reflects its multifaceted role in cellular biology, extending beyond genome maintenance [32–35].