Partha P. Kundu

Surface enhanced Raman spectroscopy of Aurora kinases: direct, ultrasensitive detection of autophosphorylation

Highly sensitive surface enhanced Raman spectroscopy (SERS) was used to study two of the tumorigenic aurora family kinases, Aurora A and Aurora B in sub-picomole quantities. Significantly, the proteins on conjugating to SERS-active citrate-capped silver nanoparticles retain their enzyme activity as demonstrated by kinase assays, making SERS suitable for studies of enzymatic processes. The ability to differentiate between two structurally similar homologous proteins further corroborates the sensitivity of this technique. Based on the available structural information we demonstrate here that SERS could be used for direct and ultra-sensitive detection of autophosphorylation. The overall reduction in SERS intensity and the presence of bands at 952 and ∼1076 cm−1 confirmed the phosphorylated state of Aurora A. Thus, the detection of autophosphorylation in a full length protein is demonstrated for the first time using SERS, which is critical to attain its active conformation.

Allosteric Transition Induced by Mg2+ Ion in a Transactivator Monitored by SERS

We demonstrate the utility of the surface-enhanced Raman spectroscopy (SERS) to monitor conformational transitions in protein upon ligand binding. The changes in proteins secondary and tertiary structures were monitored using amide and aliphatic/aromatic side chain vibrations. Changes in these bands are suggestive of the stabilization of the secondary and tertiary structure of transcription activator protein C in the presence of Mg(2+) ion, whereas the spectral fingerprint remained unaltered in the case of a mutant protein, defective in Mg(2+) binding. The importance of the acidic residues in Mg(2+) binding, which triggers an overall allosteric transition in the protein, is visualized in the molecular model. The present study thus opens up avenues toward the application of SERS as a potential tool for gaining structural insights into the changes occurring during conformational transitions in proteins.

Conformational Analysis of Molecules: Combined Vibrational Spectroscopy and Density Functional Theory Study

Vibrational spectroscopy can be broadly classified into Raman and infrared (IR). These two techniques are complementary to each other as the mechanisms behind these are different. Vibrational spectroscopy provides detail information about the structure of molecules. The advantage of this technique over X‐ray diffraction is that it can be used to probe molecules in solid, liquid or gas phase. This is especially helpful for studying biomolecules as those molecules can be probed in their physiological environment. Over the last few decades, quantum mechanical calculation has become important tool to assign bands from vibrational spectra. Combination of these two techniques has been used widely in the field of chemistry and biochemistry. In this chapter, we review some of the works that combine both of these techniques. A brief theoretical background is given for understanding the principle of these two techniques.