# Incidence of Phage Capsid Organization on the Resistance to High Energy Proton Beams
## 3 Results and Discussion  
In order to have a proper proton-solution interaction, each phage solution (200 \(\upmu\)L) in small polyethylene (PE) holders was treated according to the experimental set-up described in Figure 1.  
Since the proton path is affected by the chemical-physical features of the holder, preliminary tests with different materials were carried out to evaluate the proton energy loss before hitting the viral solution (data not shown). In our experimental conditions, the projected ion range in the solution was 321 \(\upmu\)m, thus, only 50% of the solution thickness was irradiated. To this, the solution holder was subjected to fast oscillations, which, producing liquid mixing, permitted irradiation of the entire solution during the 800 s irradiation time. Experiments were carried out in the regimes of electron energy loss and nuclear energy loss, respectively, because each phage solution was irradiated by high proton energy (4718 keV) and low energy (around 100 keV). In the first case, ionizations were mainly produced along the proton path, while in the second case, scissions, atomic collisions and the breaking of chemical bonds occurred. Figure 2a reports the SRIM simulations [40] of the proton trajectories inside the liquid solution, assumed as \"water-like\", while Figure 2b shows the stopping power trend versus the solution depth, indicating the different stopping at the target surface (electronic) and at the Bragg peak (maximal stopping at the end of ion path). From the latter, it can be deduced that the minimum stopping power is 8 keV/\(\upmu\)m, while the maximum, at the Bragg  
Figure 1: Holder made of PE, containing the phage solution (**a**), detail of the set-up adopted for the proper proton–solution interaction (**b**) and scheme of the proton energy loss in the different materials before hitting the phage solution (**c**). peak, is 40 keV / \(\upmu\)m. By considering the shape of the proton stopping power vs. depth, the average weight of its value is about 13 keV / \(\upmu\)m, which is considered to be the average linear energy transfer (LET) of the proton radiation in the irradiated solution. It is known that the proteins are prominently targeted by radiations, which induce oxidation or reduction in the functional groups exposed on the chains [41, 42]. Consequently, the resistance to radiation stress could depend on the maintenance of the capsid structure, especially against indirect damage, due to radiolytic OH\(-\) and H+ products resulting from the water radiolysis. In more complex structures, such as that of bacteriophage M13, the pVIII proteins form a tube-like structure around DNA. Specifically, each coat protein is oriented with the N-terminus, located at the outside, and the C terminus at the inside to interact with the DNA [43]. Moreover, the hydrophobic domain, located in the central part, interlocks the coat protein with those neighboring [44]. Consequently, small variations in protein subunits can affect the stability of the entire phage particle. To this purpose, two phage clones, engineered to display in pVIII capsid protein foreign peptides of 9 (P9b) and 12 amino acids (12 III1), were evaluated for their ability to maintain infectious and replicative properties after exposure to accelerated proton beams at 5.0 MeV energy, 3.8-38 pA beam current, 50-500 Gy absorbed dose and 0.0615-0.615 Gy/s dose rate (Table 1). Although the absorbed doses were high, their administration to the phage solution was given slowly (Table 1), thus, the dose rate was low, giving time to the molecular structures to reorganize and to the biological structures to partially repair the damage suffered. Phage clones were chosen on the basis of the amino acid sequence of the foreign peptide displayed on the engineered pVIII protein.