Editorial Article

Application of Infrared Probe Technique to Structural Analysis of Amyloid Fibril

Dr. Gang Ma,

Baohuan Jia, Lujuan Yang, and Gang Ma*
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China

Amyloid formation is an abnormal protein aggregation process, through which the aggregated proteins form a unique type of fibrillar aggregates termed as amyloid fibril [1]. The basic structural unit of amyloid fibril is composed of two interdigitated beta sheets.

*Corresponding author:

Gang Ma,

Amyloid formation is an abnormal protein aggregation process, through which the aggregated proteins form a unique type of fibrillar aggregates termed as amyloid fibril [1]. The basic structural unit of amyloid fibril is composed of two interdigitated -sheets with the sheets running along fibril axis and the strands running perpendicular to fibril axis. Amyloid fibril possesses several characteristic biophysical properties. It displays cross- X-ray diffraction pattern with two reflections, which are attributed to the inter-strand spacing and inter-sheet spacing in the amyloid structure, respectively. It binds to the dye Congo red and displays apple green birefringence with polarizing optics. It binds to the dye thioflavin T (ThT) and induces strong fluorescent enhancement of ThT. It usually has un-branched and straight morphology. In vivo, the deposition of amyloid fibrils in human tissues and organs is closely related to about forty serious human diseases, including some well-known disorders such as Alzheimer’s disease, mad cow disease, type II diabetes, and Parkinson’s disease [2].The close link between amyloid fibril formation and human diseases has sparked great interests among scientists worldwide to study the structure and formation mechanism of amyloid fibril.

Structural characterization of amyloid fibril is no doubt very important for the in-depth understanding of amyloid fibril formation. However, due to the semi-crystalline nature of amyloid fibril, structural characterization of amyloid fibril has been a challenging task. In recent years, infrared (IR) probe has become an emerging spectroscopic probe in elucidating protein structure and function [3,4]. The IR probe is a small IR-active organic moiety covalently attached to the side chain of one amino acid residue in a protein. The commonly used IR probes include CN, SCN, and N3. These IR probes possess two important properties. First, the stretching frequencies of these IR probes are located within a less interfered spectral window (2300 cm-1-2100 cm-1) where there is no interference of other functional groups. The only interference is due to a weak water combination mode that can be removed through proper spectral subtraction. Second, these IR probes have relatively large extinction coefficients and high dipole strengths, making them excellent probes to detect subtle structural changes within the protein.

The capability of the IR probe to provide residue-specific information makes this technique a promising technique in the application of structural analysis of amyloid fibril and mechanistic study of amyloid formation. Compared with other commonly used spectroscopic probes such as the fluorescent probe, IR probe is very small. This feature is very important when the IR probe is used in the study of amyloid fibril. As we know, amyloid fibril formation is a very delicate process. Introducing a foreign probe onto the side chain of a native protein may change the intrinsic amyloidogenic properties of these proteins if the probe is in large size, e.g. in the case of fluorescent probe. Compared with fluorescence probe, the small size of the IR probe only causes minimal perturbation to the native structure and property of the amyloidogenic protein. Second, IR probe can be used as a dual-functional structural probe. The FTIR spectrum has two important spectral regions that can be used to extract valuable structural information. The probe region provides site-specific information about the amyloid structure. In particular, it senses the interaction between the two interdigitated -sheets. In this regard, the IR probe can be considered as a quaternary structural probe for amyloid fibril. The amide I region (1700 cm-1-1600 cm-1) provides the overall secondary structural information. Different protein secondary structures have unique spectral features in this region. In particular, this region can be used to determine whether the -sheet structure of amyloid fibril is an anti-parallel or parallel -sheet structures. As we have discussed in our recent work in details, FTIR has the capability to differentiate different types of -sheet configurations [5,6]. The spectral assignment criterion is that anti-parallel -sheet features with a low-frequency component below 1640 cm-1 and a high-frequency component above 1680 cm-1 in the amide I region, while parallel -sheet structure lacks the high-frequency component above 1680 cm-1. Therefore, IR probe can be used to tackle amyloid structure at both secondary and quaternary structural levels simultaneously.

So far the appplication of IR probe in the amyloid field is still in its early stage and relevant publications are still rare [7-10]. In the following, we will briefly describe some recent work inclduing our own work regarding to the IR probe application in amyloid research. Oh et al incorporated the unnatural amino acid of -azidoalanine into a short amyloidogenic peptide of A(16-22) through solid-phase peptide synthesis (SPPS) [7]. They then demonstrated the potential use of N3 as an IR probe of the local electrostatic environment in amyloid aggregate. Marek et al introduced para-cyanophenylalanine into human islet amyloid polypeptide (IAPP) in a site-specific manner through SPPS and tackled the detailed structure of IAPP fibril [8]. Using this CN probe, they revealed clear solvent exposure differences at different sites within the fibrillar architecture. Recently, we used SCN IR probe to explore the structural details of an amyloid fibril by a 21-residue amyloidogenic peptide, A(8-28) [10]. Through a combined spectral analysis in both the probe region and the amide I region, we proposed a millipede-like structural model for A(8-28) amyloid Fibril. This structural model displays a compact central -sheet region and disordered peripheral regions, thus looking like a millipede.

In the future, we would like to see more innovative applications of IR probe in the amyloid field. Certainly we should keep in mind the limitations of IR probe technique. Unlike solid state NMR and cryo-TEM technique, an IR probe by itself cannot give a full picture of an amyloid structure. Yet, as a rapid, low cost, and easy to use technique, we believe that the IR probe can be a valuable complementary tool in future amyloid research when it is used smartly.

G. M. gratefully acknowledges the financial support from the Natural Science Foundation of Hebei Province (No. B2016201034).

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Published: 19 May 2017


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