Low-Frequency Spectroscopic Analysis of Monomeric and Fibrillar Lysozyme HIDAYATUL A. ZAKARIA, BERND M. FISCHER, ANDREW P. BRADLEY, INKE JONES, DEREK ABBOTT, ANTON P. J. MIDDELBERG, and ROBERT J. FALCONER* Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane QLD 4072, Australia (H.A.Z., A.P.J.M., R.J.F.); French-German Research Institute of Saint-Louis (ISL), 5 rue du General de Cassagnou, 68301 Saint-Louis Cedex, France (B.M.F.); School of Information Technology and Electrical Engineering, The University of Queensland, Brisbane QLD 4072, Australia (A.P.B.); and Department of Electrical and Electronic Engineering, The University of Adelaide, South Australia, SA 5005, Australia (I.J., D.A.) Terahertz time-domain spectroscopy (THz-TDS) and Fourier transform infrared (FT-IR) spectroscopy were used to generate far-infrared and low- frequency spectral measurements of monomeric lysozyme and lysozyme fibrils. The formation of lysozyme fibrils was verified by the Thioflavin T assay and transmission electron microscopy (TEM). It was evident in the FT-IR spectra that between 150 and 350 cm 1 the two spectra diverge, with the lysozyme fibrils showing higher absorbance intensity than the monomeric form. The broad absorption phenomenon is likely due to light scattered from the fibrillar architecture of lysozyme fibrils as supported by simulation of Rayleigh light scattering. The lack of discrete phonon- like peaks suggest that far-infrared spectroscopy cannot detect vibrational modes between the highly ordered hydrogen-bonded beta-pleated sheets of the lysozyme subunit. Index Headings: Terahertz; Time-domain spectroscopy; THz-TDS; Four- ier transform infrared spectroscopy; FT-IR spectroscopy; Vibrational spectroscopy; Lysozyme; Amyloid; Fibril. INTRODUCTION Despite extensive research in the area, understanding of the optical properties of proteins in the far-infrared (FIR) portion of the electromagnetic spectrum between 3 and 350 cm 1 (0.1–12 THz) is incomplete. 1 Study of the FIR spectra of proteins was initially conducted using Fourier transform infrared (FT-IR) spectroscopy with samples prepared as water-cast protein films or protein powder mixed with polyethylene pressed into pellets. 2 A broad increase in absorbance between 100 and 200 cm 1 was discovered for a range of globular proteins (including lysozyme, myoglobin, and bovine serum albumin). More recent work, conducted using Fourier transform infrared (FT-IR) analysis of the proteins lysozyme, myoglobin, and bovine serum albumin all prepared as air-dried thin films, distinguished the broad band between 100 and 200 cm 1 and showed smaller peaks centered on 320 cm 1 , 380 cm 1 , and 410 cm 1 . 3 Raman spectra of peptides had bands at around 150 and 220 cm 1 that were assigned to disulfide linkages between the peptides. 4 Studies using terahertz time-domain (THz-TDS) analysis 5–7 and FT-IR spectroscopy with a synchrotron light source 8 have focused on the frequencies below 100 cm 1 . No discrete peaks are observed below 100 cm 1 in these studies. The common theme of this research is that the major contribution to absorption of FIR radiation is the hydration levels in the protein. 6,7 The hydration level was used to indirectly detect conformational change in films of the protein bacteriorhodopsin. 9 The other key observation from this research was the similarity in the absorption spectra between 10 and 50 cm 1 (0.3–1.6 THz) of different proteins such as lysozyme and myoglobin. 4 Structured water around proteins may also be detected. 10 The differences in absorbance at 65–80 cm 1 (2.2–2.6 THz) between proteins in solution and the reference solution detected using a p-Ge laser and liquid helium detector system have been attributed to the hydration shell around the protein. 11 A recent study on b-lactoglobulin gels and polyomavirus virus-like particles illustrated potential for detecting higher-order protein structures in the spectral region of 50–450 cm 1 . 12 The vibrational modes causing the observed spectra for dry proteins between the frequencies 3 and 360 cm 1 are open for conjecture. Studies of simpler molecules related to proteins, including N-methyl acetamide 13 and the polyamides, 14–19 have assigned bands below 360 cm 1 to specific mainly torsional vibrations and to intermolecular hydrogen bonding. As polyamides are prone to forming crystalline solids, many spectra are dominated by phonon-like resonances within the crystalline lattice. While proteins are less prone to formation of crystalline structures than polyamides, several peaks were observed in the FIR spectrum of crystalline lysozyme that probably correspond to phonon-like resonances within the crystalline lattice. 20 This indicates that FIR analysis of protein samples may detect phonon-like resonances if ordered structures are present. We utilized two complementary technologies available for studying the far-infrared spectra of biological molecules: FT-IR using a liquid helium cooled bolometer with synchrotron light source and THz-TDS. Far-infrared spectroscopy has evolved over the last century 21 and has extended its capability to work at lower frequencies through the invention of the liquid- helium-cooled bolometer, Mylar beam splitters, and improved light sources such as synchrotron infrared beam lines 22 extending the frequency range down to around 20 cm 1 . The invention of THz-TDS is more recent 23 and has provided the ability to study frequencies ranging from 3 to 166 cm 1 , well below the range of conventional FT-IR spectroscopy. 24 In this paper we studied the THz/FIR spectra of native lysozyme and lysozyme fibrils created by heat treatment at an acidic pH. 25 Native lysozyme is monomeric and has structure that includes alpha helices. Fibrils are formed by partially unfolded lysozyme molecules, which bind together with multiple hydrogen bonds to form intermolecular b-pleated sheets. Chains of these linked protein subunits form linear structures or protofilaments. 26 These protofilaments then twist around each other to form multi-chain fibrils. Fibrils are highly ordered protein structures and are obvious candidates for Received 15 October 2010; accepted 14 December 2010. * Author to whom correspondence should be sent. E-mail: r.falconer@uq. edu.au. DOI: 10.1366/10-06162 260 Volume 65, Number 3, 2011 APPLIED SPECTROSCOPY 0003-7028/11/6503-0260$2.00/0 Ó 2011 Society for Applied Spectroscopy