RESUMO
UV resonance Raman (UVRR) spectroscopy is a powerful tool for investigating the structure of biological molecules, such as proteins. Numerous UVRR spectroscopic markers that provide information on the structure and environment of the protein backbone and of amino acid side chains have recently been discovered. Combining these UVRR markers with hydrogen-deuterium exchange and advanced statistics is a powerful tool for studying protein systems, including the structure and formation mechanism of protein aggregates and amyloid fibrils. These techniques allow crucial new insights into the structure and dynamics of proteins, such as polyglutamine peptides, which are associated with 10 different neurodegenerative diseases. Here we summarize the spectroscopic structural markers recently developed and the important insights they provide.
Assuntos
Amiloide/análise , Muramidase/análise , Peptídeos/análise , Análise Espectral Raman/métodos , Sequência de Aminoácidos , Teorema de Bayes , Humanos , Marcação por Isótopo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Análise Espectral Raman/instrumentação , Termodinâmica , VibraçãoRESUMO
Deep UV resonance Raman spectroscopy is a powerful technique for probing the structure and formation mechanism of protein fibrils, which are traditionally difficult to study with other techniques owing to their low solubility and noncrystalline arrangement. Utilizing a tunable deep UV Raman system allows for selective enhancement of different chromophores in protein fibrils, which provides detailed information on different aspects of the fibrils' structure and formation. Additional information can be extracted with the use of advanced data treatment such as chemometrics and 2D correlation spectroscopy. In this chapter we give an overview of several techniques for utilizing deep UV resonance Raman spectroscopy to study the structure and mechanism of formation of protein fibrils. Clever use of hydrogen-deuterium exchange can elucidate the structure of the fibril core. Selective enhancement of aromatic amino acid side chains provides information about the local environment and protein tertiary structure. The mechanism of protein fibril formation can be investigated with kinetic experiments and advanced chemometrics.
Assuntos
Amiloide/química , Agregação Patológica de Proteínas/genética , Análise Espectral Raman/métodos , Sequência de Aminoácidos/genética , Amiloide/genética , Conformação Proteica , Dobramento de Proteína , Raios UltravioletaRESUMO
The vibrational circular dichroism (VCD) spectra of microcrystals of fibril-forming peptides have been measured for the first time. VCD spectra were measured and compared for microcrystals and fibrils formed from the same peptide, human islet amyloid polypeptide (IAPP, amylin). Structural information related to the supramolecular chirality of both the microcrystals and the fibrils, as well as the VCD enhancement mechanisms in fibrils and microcrystals, is obtained from these spectral comparisons. It is concluded that strongly enhanced VCD does not require braiding of two or more filaments that is permitted in fibrils but not microcrystals.