International Journal of Biological Macromolecules 94 (2017) 634–641 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Biophysical characterization of soluble Pseudomonas syringae ice nucleation protein InaZ fragments Yu Jin Han a,1 , HyoJin Song a,1 , Chang Woo Lee b,c , Nguy ˜ ˆ en Hoàng Ly d , Sang-Woo Joo d , Jun Hyuck Lee b,c , Soon-Jong Kim e , SangYoun Park a,∗ a School of Systems Biomedical Science, Soongsil University, Seoul 06978, Republic of Korea b Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea c Department of Polar Sciences, Korea University of Science and Technology, Incheon 21990, Republic of Korea d Department of Chemistry, Soongsil University, Seoul 06978, Republic of Korea e Department of Chemistry, Mokpo National University, Muan 58554, Republic of Korea a r t i c l e i n f o Article history: Received 28 June 2016 Received in revised form 21 September 2016 Accepted 18 October 2016 Available online 20 October 2016 Keywords: Ice nucleation protein Bacterial ice nuclei InaZ Pseudomonas syringae a b s t r a c t Ice nucleation protein (INP) with its functional domain consisting of multiple 48-residue repeat units effectively induces super-cooled water into ice. Circular dichroism and infrared deconvolution analyses on a soluble 240-residue fragment of Pseudomonas syringae InaZ (InaZ240) containing five 48-residue repeat units indicated that it is mostly composed of -sheet and random coil. Analytical ultracentrifuga- tion suggested that InaZ240 behaves as a monomer of an elongated ellipsoid. However, InaZ240 showed only minimum ice binding compared to anti-freeze proteins. Other P. syringae InaZ proteins with more 48-residue repeat units were made, in which the largest soluble fragment obtainable was an InaZ with twelve 48-residue repeat units. Size-exclusion chromatography analyses further suggested that the over- all shape of the expressed InaZ fragments is pH-dependent, which becomes compact as the numbers of 48-residue repeat unit increase. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Water in a metastable state resists ice formation to a super- cooled state well below the 0 ◦ C freezing point. The research field of bacterial ice nucleation was established when Pseudomonas syringae bacteria on the surface of agricultural plants were found to function as ice crystal nucleating centers [1]. This function in P. syringae was attributed to ice nucleation protein (INP) which is a membrane protein at the outer membrane [2–4]. INP induces ice formation by acting as ice crystal nuclei (seeds) for super-cooled water molecules to assemble, which triggers water freezing into ice. Because most plants lack tolerance in intracellular ice formation, Abbreviations: AUC, analytical ultracentrifugation; AFP, anti-freeze protein; CD, circular dichroism; FPLC, fast protein liquid chromatography; INP, ice nucleation protein; IR, infrared; LeIBP, Leucosporidium ice-binding protein; Ni-NTA, Ni 2+ - nitrilotriacetic acid; NMR, nuclear magnetic resonance; RMS, root-mean-square; RPM, rotation per minute; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC, size-exclusion chromatography. ∗ Corresponding author at: School of Systems Biomedical Science, Soongsil Uni- versity, 369 Sangdo-ro, Dongjak-gu, Seoul 06978, Republic of Korea. E-mail address: psy@ssu.ac.kr (S. Park). 1 These authors contributed equally to this work. bacteria with ice nucleation capability bring severe frost damage to the plants [5–7]. Among the many INPs, the protein product of P. syringae inaZ gene was the first to be characterized in detail [3,8,9]. The struc- ture of InaZ is divided into an N-terminal domain (175 residues), a central domain (∼1000 residues), and a C-terminal domain (49 residues). While the N- and C-terminal domains function to anchor the protein to the outer membrane of the bacteria, the central domain which contains the highly repetitive sequences is respon- sible for the functional ice nucleation activity of InaZ (Fig. 1). The central repetitive domain consists of twenty-five 48-residue repeat units, in which the repeat units themselves contain three blocks of 16-residue repeat (AGYGSTxTAxxxSxLx, x is a non-conserved residue) (Fig. 1). Theoretical models of this central repetitive domain suggested that it folds into planar arrays of anti-parallel - sheets [10] or to a -helix [11,12]. However, either X-ray crystal or NMR spectroscopy structures which can elaborate on the ice nucle- ation mechanism have not yet been provided. Since, both structural determination techniques require milligram amounts of protein, the lack of structure can be partly due to the limited amounts of InaZ proteins obtainable. Other than the studies performed on chemically synthesized peptides of InaZ [13,14], an attempt to make highly soluble InaZ http://dx.doi.org/10.1016/j.ijbiomac.2016.10.062 0141-8130/© 2016 Elsevier B.V. All rights reserved.