Crystals from Light:  Photochemically Induced Nucleation of Hen Egg-White Lysozyme

Abstract

In this paper, we demonstrate that the irradiation of a metastable supersaturated lysozyme solution by a light produced by a continuous 150 W Xe lamp induces nucleation and that the mechanism is photochemically induced by a one-photon process. The irradiation of lysozyme molecules produces residual tryptophan radicals, which enhance the attractive interactions between molecules in solution, as shown by dynamic light scattering measurements, and increase the nucleation rate of lysozyme. When the intermediate is excited by visible light, the intermediate is converted into a permanently damaged species, and light-induced nucleation is inhibited, as shown by scavenger experiments (irradiation with two colors of excitation). These results suggest a new method for controlling nucleation and growth from metastable solutions that could be use in structural genomics and the pharmaceutical industry, for instance.

Influence of energy and wavelength on femtosecond laser-induced nucleation of protein

“…The photochemical covalent-bonded dimer of HEWL is considered to enhance the nucleation because dimer formation by weak physical interactions (van der Waals force or hydrogen bond) is energetically unfavorable [13][14][15]. In our system, however, contribution of the dimers on nucleation is rather small, because the enhancement of nucleation by the laser irradiation of k = 260 nm was same as that of k = 780 nm (Figure 3c,f and Figure 4).…”

“…However, the intensities of HEWL dimer band at 28 kDa increased. Okutsu et al reported that UV light irradiation produces the photochemical covalent-bonded protein dimers, which enhance nucleation [13][14][15]. Figure 5b shows the closeup of the bands at 28 kDa and band intensity.…”

“…The generation of such insoluble aggregate will lead to decrease in HEWL concentration, i.e., a reduction in supersaturation. Since a femtosecond laser has a higher peak intensity [$GW] than a Xe lamp [$100 W], proteins easily undergo the simultaneous and/or stepwise multiphoton absorption, which can produce the permanent damaged species [13][14][15].…”

“…In contrast, the laser irradiation at k = 260 nm produced white turbidity at the focal point (Figure 2b). Since no white turbidity was formed in the solution without NaCl (Figure 2c and d), the white turbidity is thought to be aggregates of HEWL [14]. Tetragonal single crystals of HEWL were observed at 48 h after the laser irradiation.…”

“…To date, light-induced nucleation has been demonstrated by optical Kerr effect [1][2][3][4][5][6][7][8], photon pressure [9,10], and photochemical reactions [11][12][13][14][15][16]. As another approach, we have proposed the focused irradiation of a femtosecond laser [17][18][19][20][21][22][23][24], whose peak intensity is $TW/cm 2 .…”