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Circular dichroism ( CD ) spectroscopy belongs to the family of chiroptical methods. These methods utilize the interaction of circularly polarized light ( CPL ) with chiral molecules and molecular systems to obtain information about their structure and electronic or vibrational states. A CD spectrum Δϵ(ν) is the difference of two absorbance spectra: one measured with left circularly polarized light ( LCPL ) and the other one recorded with right circularly polarized light ( RCPL ) Δϵ(ν) = ϵ L (ν)–ϵ R (ν). CD spectra can be measured as electronic circular dichroism ( ECD ) in spectral regions of electronic transitions ( ultraviolet ( UV ) and visible ( VIS ) light) and as vibrational circular dichroism ( VCD ) in the infrared ( IR ) spectral regions. An ECD spectrum for chiral fluorophores can be also recorded as the difference excitation spectrum for fluorescence spectra excited by LCPL and RCPL, respectively ( fluorescence‐detected circular dichroism ( FDCD )). Raman optical activity ( ROA ) and circularly polarized luminescence spectroscopies complete the family of currently developed chiroptical methods. The differences Δϵ(ν) measured as CD are typically ∼10 −5 of sample absorbance. Special modifications of dispersive (for ECD and VCD) or Fourier transform infrared ( FTIR ) (for VCD) spectrometers are needed to measure CD with a reasonable signal‐to‐noise ratio ( S/N ). Polarization modulation of the incident light by a photoelastic modulator and synchronous electronic processing of the resulting photoelectric signal in the spectrometers are typically used for this purpose. Molecular structure is encoded in the CD spectra because the chiral field of the CPL wave that induces a spectral transition in the chiral molecule can be observably altered both by the transition electron density redistribution (as in conventional absorption spectroscopy) and by the transition magnetic field accompanying the molecular charge redistribution. The structure and conformation of the studied molecule define the relative orientation of the characteristic directions of these two effects. This relative orientation affects the probability of absorption of photons of RCPL and LCPL and, in turn, determines the CD sign, the primary information that is unique for CD spectroscopy. In the absolute value, CD intensity (“secondary” unique information) is related to the angle of electric and magnetic transition moments and can, therefore, be converted into the molecular conformation once a suitable theoretical model is available. For complicated molecules (biomolecules, proteins, nucleic acids) where the corresponding theoretical calculations are too complex, empirical interpretive methods based on reference sets of spectra measured for molecules with known structures (from X‐ray or nuclear magnetic resonance ( NMR ) or cryo‐electron microscopy experiments) are used with success. For example, using these empirical methods, the fractions of regular secondary structures ( secondary structure fraction ( SSF )) in globular proteins can be determined with a relative error of 3–7% and the number of secondary structure segments in proteins with a typical error of one to three segments per protein fold. CD spectroscopy is highly sensitive to polarization artifacts that can be related to optical imperfections of the optical parts of the spectrometer. To achieve an acceptable S/N, the sample total absorbance A should be also controlled (typically A < 1). This, in effect, restricts the selection of usable solvents and largely determines the concentration and/or path length ranges of studied samples.
Circular dichroism ( CD ) spectroscopy belongs to the family of chiroptical methods. These methods utilize the interaction of circularly polarized light ( CPL ) with chiral molecules and molecular systems to obtain information about their structure and electronic or vibrational states. A CD spectrum Δϵ(ν) is the difference of two absorbance spectra: one measured with left circularly polarized light ( LCPL ) and the other one recorded with right circularly polarized light ( RCPL ) Δϵ(ν) = ϵ L (ν)–ϵ R (ν). CD spectra can be measured as electronic circular dichroism ( ECD ) in spectral regions of electronic transitions ( ultraviolet ( UV ) and visible ( VIS ) light) and as vibrational circular dichroism ( VCD ) in the infrared ( IR ) spectral regions. An ECD spectrum for chiral fluorophores can be also recorded as the difference excitation spectrum for fluorescence spectra excited by LCPL and RCPL, respectively ( fluorescence‐detected circular dichroism ( FDCD )). Raman optical activity ( ROA ) and circularly polarized luminescence spectroscopies complete the family of currently developed chiroptical methods. The differences Δϵ(ν) measured as CD are typically ∼10 −5 of sample absorbance. Special modifications of dispersive (for ECD and VCD) or Fourier transform infrared ( FTIR ) (for VCD) spectrometers are needed to measure CD with a reasonable signal‐to‐noise ratio ( S/N ). Polarization modulation of the incident light by a photoelastic modulator and synchronous electronic processing of the resulting photoelectric signal in the spectrometers are typically used for this purpose. Molecular structure is encoded in the CD spectra because the chiral field of the CPL wave that induces a spectral transition in the chiral molecule can be observably altered both by the transition electron density redistribution (as in conventional absorption spectroscopy) and by the transition magnetic field accompanying the molecular charge redistribution. The structure and conformation of the studied molecule define the relative orientation of the characteristic directions of these two effects. This relative orientation affects the probability of absorption of photons of RCPL and LCPL and, in turn, determines the CD sign, the primary information that is unique for CD spectroscopy. In the absolute value, CD intensity (“secondary” unique information) is related to the angle of electric and magnetic transition moments and can, therefore, be converted into the molecular conformation once a suitable theoretical model is available. For complicated molecules (biomolecules, proteins, nucleic acids) where the corresponding theoretical calculations are too complex, empirical interpretive methods based on reference sets of spectra measured for molecules with known structures (from X‐ray or nuclear magnetic resonance ( NMR ) or cryo‐electron microscopy experiments) are used with success. For example, using these empirical methods, the fractions of regular secondary structures ( secondary structure fraction ( SSF )) in globular proteins can be determined with a relative error of 3–7% and the number of secondary structure segments in proteins with a typical error of one to three segments per protein fold. CD spectroscopy is highly sensitive to polarization artifacts that can be related to optical imperfections of the optical parts of the spectrometer. To achieve an acceptable S/N, the sample total absorbance A should be also controlled (typically A < 1). This, in effect, restricts the selection of usable solvents and largely determines the concentration and/or path length ranges of studied samples.
Circular dichroism ( CD ) is the difference in absorption, , of left and right circularly polarized light: For randomly oriented systems such as solutions, only chiral molecules will show any CD intensity corresponding to their absorption bands. Chiral is derived from the Greek word χϵιρ meaning hand and describes something that cannot be superposed on its mirror image by rotation. Two molecules that are mirror images of each other are often referred to as enantiomers and equimolar mixtures of two enantiomers form a racemic mixture which has no net CD intensity in solution. CD can be used to analyze chiral structures, such as a protein secondary structure, and to probe interactions between chiral molecules and other molecules. Linear dichroism ( LD ) is the difference in absorption of light linearly polarized parallel and perpendicular to an orientation axis: LD can be used to provide orientation information about subunits of a molecular system such as small molecules absorbed onto stretched films, flow‐oriented DNAs and fibrous proteins, and lipid bilayer systems. Both circular and LD are absorbance techniques and most CD instruments will also measure LD. Both CD and LD can also be enhanced by fluorescence detection with either incident circularly or linearly polarized light (fluorescence detected circular and linear dichroism) and/or measurement of the circular or linear nature of the emitted light.
Data analysis and manipulation software are vulnerable to user error during data processing and computations take considerable time when handling huge data and multiple repetitive tasks. These problems are usually mitigated by creating an app to repeat any given task reproducibly any number of times. This paper discusses the development of app that systematically automates the <i>ad hoc</i> approach for derandomization of proteins and, or peptides. Thirty second-year undergraduates with little-to-no prior knowledge of computer programming are (were) asked to create this app with modules that sequentially convert spectra from original units to molar extinction and subtract baseline spectrum from the resultant spectra, derandomize the spectra by removing suspected significant unfolded domains from them, concatenate the generated files to a single file in an acceptable format for structural analysis, process our group structural algorithm output files into a user-friendly format to ease data analysis. In addition, they are (were) asked to prepare protein solution, determine its concentration spectroscopically, collect circular dichroism measurements of the protein, derandomize the protein spectra, and determine the secondary structure of the resultant protein spectra with our structure algorithm. The assessment results demonstrated that the students could prepare samples for CD analysis, collect spectra of proteins, and create an app to automate the <i>ad hoc</i> approach. The hands-on activities enable students to acquire knowledge in basic programming and circular dichroism, CD spectroscopy.
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