Applied Bioinformatics Week 11. Topics Protein Secondary Structure RNA Secondary Structure.
Determining Protein Secondary Structure with …...Determining Protein Secondary Structure with...
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Introduction Although crystallography and NMR are the primary tools for determining geometrical protein structures there are significant limitations in the range of proteins that these techniques can address. FT-IR gives less detailed information but it has some important advantages. It can be applied to any protein, it requires a relatively small sample, and the measurements can be made in solution. The applications for FT-IR include determining secondary structure of novel proteins, identifying conformations in formulations, measuring the kinetics of conformational changes with temperature or other perturbations, and investigating protein-ligand interactions. The information about secondary structure is contained in the shape of the amide-1 band in the IR spectrum. This requires careful measurement as it overlaps with the water band at 1640cm -1 and water vapor absorptions. Both transmission and ATR techniques can be used, but adsorption of the protein on to the crystal surface is a potential problem with ATR. For transmission measurements pathlengths between 6 and 10μm are generally used. The secondary structure is analyzed as a mixture of different amounts of various sub-structures such as α-helix and β-sheet, each of which has characteristic absorptions. In curve-fitting and deconvolution approaches the amide-1 band is analyzed explicitly as the superposition of such bands. Chemometric approaches use a library of spectra from proteins of known secondary structure. Determining Protein Secondary Structure with Spectrum™ 100 FT-IR Spectroscopy APPLICATION BRIEF Author RichardSpragg PerkinElmer, Inc. Chalfont Road Seer Green Beaconsfield Buckinghamshire, UK
Transcript of Determining Protein Secondary Structure with …...Determining Protein Secondary Structure with...
Introduction
Although crystallography and NMR are the primary tools for determining geometrical protein structures there are significant limitations in the range of
proteins that these techniques can address. FT-IR gives less detailed information but it has some important advantages. It can be applied to any protein, it requires a relatively small sample, and the measurements can be made in solution. The applications for FT-IR include determining secondary structure of novel proteins, identifying conformations in formulations, measuring the kinetics of conformational changes with temperature or other perturbations, and investigating protein-ligand interactions.
The information about secondary structure is contained in the shape of the amide-1 band in the IR spectrum. This requires careful measurement as it overlaps with the water band at 1640cm-1 and water vapor absorptions. Both transmission and ATR techniques can be used, but adsorption of the protein on to the crystal surface is a potential problem with ATR. For transmission measurements pathlengths between 6 and 10μm are generally used.
The secondary structure is analyzed as a mixture of different amounts of various sub-structures such as α-helix and β-sheet, each of which has characteristic absorptions. In curve-fitting and deconvolution approaches the amide-1 band is analyzed explicitly as the superposition of such bands. Chemometric approaches use a library of spectra from proteins of known secondary structure.
Determining Protein Secondary Structure with Spectrum™ 100
FT-IR Spectroscopy
A P P L I C A T I O N B R I E F
Author
�Richard�SpraggPerkinElmer, Inc.Chalfont RoadSeer GreenBeaconsfieldBuckinghamshire, UK
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Experimental
At a concentration of 4mg/mL in a 6μm pathlength cell the absorbance of the amide-1 band of a protein is typically about 0.005A, which corresponds to a difference in transmission between sample and buffer of 0.3%. In measuring these small signals the temperature dependence of the water absorption and interference from water vapor are more significant than the signal-to-noise performance of the spectrometer.
Proteins were measured in water at concentrations of 20 and 4mg/mL. The instrument was a Spectrum 100 with DTGS detector, using a sample shuttle to minimize atmospheric interference. With 64 scans at 4cm-1 the measurement time for background and sample together was less than 15 minutes. The temperature was not controlled but the cell was allowed to stabilize in the sample compartment.
The data shown here were obtained with a Biocell™ transmission cell (BioTools Inc., Jupiter, Florida www.btools.com). This consists of two calcium fluoride windows, one of which has a recess providing a pathlength of about 6μm. Filling is very easy in comparison to a conventional sealed cell with such a short pathlength. A few microliters of solution are placed in the recess and any excess is displaced into a groove when the top window is put in place. The pathlength can be adjusted over a narrow range by varying the pressure between the windows. This allows the water absorbance of solution and buffer to be matched which aids subtraction.
The secondary structure analysis used the Prota™ software from BioTools. This is a factor analysis approach based on a library of fifty proteins of known secondary structure.
Results
Figure 1. Overlaid transmission spectra of water and BSA at 4mg/mL. 64 scans at 4cm-1, DGTS.
The secondary structure analysis is based on Amide-1 and Amide-2 bands, using the region 1800 to 1400cm-1 after subtraction of the water absorption.
Figure 2. Amide-1 and -2 bands of BSA. Amide-1 absorbance 0.026 at 20mg/mL, 0.005 at 4mg/mL.
Secondary�structure�calculations
BSA� Helix� Sheet� Bend� Turn� Coil� Sum
20mg/mL 56.35 4.05 7.61 12.18 23.80 103.98
4mg/mL 61.64 0.42 7.68 11.79 23.96 103.93
Summary
FT-IR spectra can provide detailed information about the secondary structure of proteins in solution. While these are challenging measurements the stability and exceptional signal-to-noise performance of the Spectrum 100 makes them rapid and routine.
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