Techniques for studying protein primary structure
Structural analysis of a protein usually starts with the determination of its primary structure: the amino acid sequence. This can rely on two types of methods: 1) mass spectrometry (MS)-based protein sequencing, and 2) Edman degradation. The former is particularly useful to generate full sequences of large proteins (more than 40 amino acid residues). Nowadays, the advancement of MS-based proteomics has led to the creation of de novo protein sequencing, allowing for rapid and accurate sequence determination of any given protein. The primary structure of a protein can also be analyzed by means of peptide mapping, another MS-based method well suited for verifying sequence and identifying sequence variations like point mutations.
Techniques for studying protein secondary structure
To study a protein’s secondary structure, the most common method is circular dichroism spectroscopy (CD). It measures different secondary structural elements in a protein, including α helix and β sheet, and random coil, based on their characteristic spectra in the far-UV region of the spectrum. These spectra can then be used to quantify the fraction of each secondary structural element in the protein. Another biophysical method, Fourier transform infrared (FTIR) spectroscopy, can also be used to estimate secondary-structural components in a protein by measuring the wavelength and intensity of infrared radiation (IR) absorption by a protein sample. Additionally, there are a variety of bioinformatics tools for secondary structure prediction that researchers can benefit from to interpret the protein structural organization.
Techniques for studying protein tertiary structure
The tertiary structure, or the three-dimensional structure, of a protein, is mainly determined using X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryogenic electron microscopy (cryo-EM). Each method has its advantages and disadvantages. Generally, with these methods, different types of experimental data (i.e., X-ray diffraction, chemical shift) are gathered to measure distances between atoms and yield a three-dimensional model of the protein structure at the atomic level. To date, the 3D structures of more than 190,000 different proteins have been determined. In addition to the above methods, hydrogen-deuterium exchange (HDX)-MS is a powerful analytical technique often combined with other structural techniques to obtain important information on protein conformation, dynamics, and folding, therefore a more comprehensive picture. Certainly, a number of in silico tools have been rapidly emerging in recent years for the prediction of a protein’s 3D structure, such as Alphafold.
Techniques for studying protein quaternary structure
Finally, the quaternary structure of a protein can be studied using the same methods for the tertiary structure. In particular, the advancement of cryo-EM is gaining popularity for high-resolution visualization of large protein complexes with complicated quaternary structures. HDX-MS is also particularly useful for studying protein interactions, such as antibody-antigen binding.
Summary of protein structure study methods
Table of methods that can be used to study each level of protein structure
Rapid Novor provides multiple services that can facilitate the analysis and characterization of protein structure. Please visit our services pages—de novo protein sequencing, peptide mapping, and HDX-MS—to learn more about these technologies, and reach out to our scientists for inquiries.