Secondary Structure Analysis

Protein nanocages have attracted wide attention from researchers as candidates in medical uses because of the advantageous structure and property features. Most of the protein nanocages are natural proteins. In the development of protein nanocages as drug delivery platforms or vaccines, the structures of the natural protein nanocages are supposed to be maintained. However, the usage of expression hosts with different protein folding systems to produce recombinant protein nanocages and the introduction of primary sequence alteration through mutations or fusions may cause structural alterations. Protein secondary structure is a reliable predictor of protein higher-order structures, and it is closely related to protein biochemical properties, such as hydrophobicity and stability. The determination of secondary structure can be a rapid and effective approach to get structural and property information about designed protein nanocages.

Secondary structure in protein refers to the 3-dimension structures of local segments of proteins formed by the non-covalent interactions between atoms in the protein backbone. Hydrogen bonds between the carbonyl O of one amino acid and the amino H of another are the main interactions, whilst interactions between residue side-chain atoms are not involved. α-helices and β-sheet are two common secondary structures. β-turn and Ω loop occur in fewer cases. Other unstructured areas are called random coils.

Fig 1. Hydrogen Bond Patterns in β-sheet (A) and α-helix (B) (Friedrich, D., et al., 2020)
Nitrogen atoms are shown as blue, oxygen as red, hydrogen atoms as small white, and carbon atoms as big white spheres.

Creative BioMart Nanocage is experienced in far UV circular dichroism (CD ) and Fourier transform infrared spectrometry (FTIR) for protein nanocage secondary structure analysis. Our service includes sample pretreatment, sample preparation, sample detection, and result analysis.

1. Far-UV Circular Dichroism Spectrometry

Mechanism: Asymmetric molecules absorb right- and left-handed circularly polarized light to a different extent. In far UV CD spectrometry, the asymmetric secondary structures demonstrate different characteristics. For example, α-helix shows two negative bands at approximatly 208 and 222 nm and a positive band at 193 nm. β-sheet demonstrates a negative band near 216 nm, a positive band between 195 and 200 nm, and a negative band near 175 nm.

Sample requirements: Protein nanocages in solution or freeze-dried protein nanocages. Chloride salts and additives that absorb far UV or can be activated by far UV should be strictly avoided.

Sample pretreatments: Protein concentration determination, buffer exchange, protein concentration, and protein purification.

Applications: Quantitative determination of protein nanocage secondary structure and protein secondary structure stability evaluation. Protein secondary structure stability can be performed at different temperatures and/or with the presence of different concentrations of denaturants such as urea.

2. Fourier Transform Infrared Spectrometry

Mechanism: Infrared spectrometry detects the vibration of chemical bonds. In protein infrared spectra, Amide I and Amide II bands are associated with protein secondary structure. C=O stretching forms Amide I band (1600-1700 cm^-1), whereas N—H bending forms Amide II. Amide I band is used more frequently in secondary structure analysis than Amide II. Quantitative analysis is achieved through the deconvolution of the Amide I band into multiple peaks corresponding to different secondary structures.

Sample requirements: Protein nanocage powders or protein nanocage in solution.

Sample pretreatment: Protein concentration.

Application: Protein nanocage quantitative secondary structure determination.

If you would like to determine your protein nanocage secondary structure, please feel free to contact us through online inquiry.

References

• Friedrich, D., et al. (2020). "MAS NMR detection of hydrogen bonds for protein secondary structure characterization." Journal of biomolecular NMR, 1-10.
• Greenfield, N. J. (2006). "Using circular dichroism spectra to estimate protein secondary structure." Nature protocols, 1(6), 2876.
• Pelton, J. T., Larry R. M. (2000). "Spectroscopic methods for analysis of protein secondary structure." Analytical biochemistry, 277(2),167-176.