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Related Concept Videos

Infrared (IR) Spectroscopy: Overview01:09

Infrared (IR) Spectroscopy: Overview

When electromagnetic radiation passes through a material, atoms or molecules transition from a lower to a higher energy state by absorbing radiation corresponding to the energy difference between the two states. The absorption of infrared (IR) radiation causes transitions between vibrational energy levels in a molecule. Therefore, IR spectroscopy is a useful analytical tool for determining the molecular structure of molecules.
Different compounds display unique properties due to their...
IR Spectrometers01:25

IR Spectrometers

There are two main infrared (IR) spectrophotometers: dispersive IR spectrometers and Fourier transform infrared (FTIR) spectrometers. In a dispersive IR spectrometer, a beam of infrared radiation produced by a hot wire is divided into two parallel equal-intensity beams using mirrors. One beam passes through the sample, while another is a reference beam. The beams then move through the monochromator, which separates the radiations into a continuous spectrum of different frequencies. The...
Applications of IR Spectroscopy: Overview01:11

Applications of IR Spectroscopy: Overview

The non-destructive nature and ability to provide valuable chemical information make IR spectroscopy a versatile technique with broad applications in various scientific and industrial fields. IR spectroscopy is commonly used to identify and characterize organic and inorganic compounds. It provides information about the functional groups present in a molecule and the bonding between atoms. This helps in the structural elucidation of compounds during organic synthesis, pharmaceutical research,...
IR Spectroscopy: Molecular Vibration Overview01:24

IR Spectroscopy: Molecular Vibration Overview

When Infrared (IR) radiation passes through a covalently bonded molecule, the bonds transition from lower to higher vibrational levels. The fundamental vibrational motions that result in infrared absorption can be classified as stretching or bending vibrations.
Stretching vibrations are vibrational motions that occur along the bond line, changing the bond length or distance between two bonded atoms. They are further distinguished as symmetric or asymmetric. In symmetric stretching, the...
IR Spectrum01:19

IR Spectrum

When infrared (IR) radiation passes through a molecule, the bonds stretch or bend by absorbing the radiation. This absorption creates the molecule's absorption spectrum, which is the plot of its percentage transmittance versus wavenumber.
Transmittance is defined as the ratio of the radiant power passing through a sample to that from the radiation's source. Multiplying the transmittance by 100 gives the percent transmittance (%T), which varies between 100% (no absorption) and 0% (complete...
Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview01:13

Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview

Attenuated total reflectance (ATR) infrared spectroscopy is a powerful analytical technique used to study the composition of materials. It is widely employed in chemistry, materials science, forensic science, and other fields where sample characterization is required. ATR has several advantages over traditional transmission IR spectroscopy, including the requirement of little to no sample preparation and the ability to analyze a wide range of samples.
The ATR process begins by directing a beam...

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Optical Photothermal Infrared-Fluorescence In Situ Hybridization (OPTIR-FISH)
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Isotope-edited infrared spectroscopy.

Ginka S Buchner1, Jan Kubelka

  • 1Department of Chemistry, University of Wyoming, Laramie, WY, USA.

Methods in Molecular Biology (Clifton, N.J.)
|July 5, 2012
PubMed
Summary
This summary is machine-generated.

Isotope-edited infrared spectroscopy uses carbon-13 labeling to reveal protein structure and dynamics at specific sites. This method analyzes amide I

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Area of Science:

  • Biophysical Chemistry
  • Spectroscopy
  • Protein Science

Background:

  • Isotope-edited infrared (IR) spectroscopy offers site-specific insights into protein structure and dynamics.
  • Labeling with carbon-13 ((13)C) on amide carbonyls generates sidebands in amide I' vibrations.
  • These sidebands provide information on local conformation and solvent exposure without perturbing protein structure.

Purpose of the Study:

  • To detail the methodology for synthesizing (13)C isotopically edited protein samples.
  • To outline experimental IR spectroscopic measurements for these samples.
  • To explain the analysis of site-specific structural changes using thermal unfolding IR data.

Main Methods:

  • Chemical synthesis of proteins incorporating specific isotopically labeled amino acids.
  • Isotope editing using carbon-13 ((13)C) labeling at amide carbonyl sites.
  • Infrared (IR) spectroscopy, specifically analyzing amide I' vibrations and sidebands.
  • Thermal unfolding experiments to assess protein structural changes.

Main Results:

  • Successful synthesis of (13)C isotopically edited protein samples.
  • Detection of characteristic sidebands in amide I' vibrations due to (13)C labeling.
  • Correlation of spectral changes with site-specific conformational and solvent exposure information.
  • Quantification of structural alterations during thermal denaturation.

Conclusions:

  • Isotope-edited IR spectroscopy is a robust technique for site-specific protein structural and dynamical studies.
  • The described methods enable the preparation and analysis of labeled proteins.
  • This approach provides valuable, non-perturbing insights into protein behavior, particularly during thermal unfolding.