PIKE Technologies, Inc.

Protein secondary structure during thermal unfolding and aggregation is readily acquired using IR spectroscopy and a temperature-controlled mid-IR transmission accessory. Myoglobin was used as a model system to illustrate the method.

	FT-IR spectroscopy is a powerful tool for measuring protein secondary structure. Using a temperature-controlled transmission accessory, the evolution of protein secondary structure through thermal transitions is readily accessible. In fact, the experiment and analysis are relatively quick; therefore, a set of proteins can be rapidly screened for stability and temperature-dependent conformational changes. This information is particularly important when evaluating multiple protein mutants.

	To demonstrate the simplicity of secondary structure evaluation using FT-IR spectroscopy, the well-characterized protein, myoglobin (1), was unfolded in D

O (3.5 µM, 50 µm pathlength) from 25 °C to 90 °C in 5 °C increments using the Falcon Mid-IR Transmission Accessory from PIKE Technologies, Inc. The accessory’s Peltier elements allow for quick ramp rates and temperatures ranging from 5 °C to 130 °C. Temperature profile and spectral data collection was executed by PIKE TempPRO

 software. The amide I band was deconvolved by Fourier deconvolution (bandwidth 50 cm

, enhancement 2) and frequencies were obtained from second derivative spectra.

	Four peaks dominate the 25 °C spectrum. The largest peak at 1649 cm

 is assigned to α-helix and random coil structure. From the structure obtained by X-ray diffraction (Figure 1 inset), myoglobin is comprised of several α-helix and random coil regions (2). Two other peaks are observed at 1630 and 1679 cm

 and correspond to extended chain and turn regions, respectively (1). The last peak at 1612 cm

 is not amide I vibration but instead tyrosine side chain absorption (3). Thus, from quick spectral manipulation and inspection, the secondary structure at 25 °C is readily determined.

					Figure 1: FT-IR spectra of myoglobin between 25 °C and 90 °C. Myoglobin is rendered with UCSF Chimera (4).

	As the temperature increases, significant changes in the myoglobin secondary structure are observed. Between 25 °C and 70 °C, the 1649 cm

 peak height decreases and linewidth increases. These changes in the amide I band reflect increased protein structural disorder. A temperature increase from 70 °C to 75 °C causes a drop in the 1649 cm

 band intensity. Simultaneously, two peaks grow in at 1683 and 1637 cm

. These changes indicate conversion of random coil/α-helix to intermolecular β-sheet. Further conversion to β-sheet is observed as the temperature rises to 90 °C. The final β-sheet structure is an insoluble aggregate. This aggregation process is not reversed when the temperature is driven back to 25 °C.

	Using the PIKE Falcon accessory makes monitoring the thermal conversion of myoglobin from α-helix to random coil to β-sheet simple.

		F. Meersman, L. Smeller, and K. Heremans, 

	6125 Cottonwood Drive, Madison, WI 53719

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Simple Method for Monitoring Protein Secondary Structure During Thermal Unfolding and Aggregation

Yeast suspensions were dried and analyzed via FT-IR using an automated transmission sampling accessory. Up to six-fold sampling capacity increase may be realized through automation. 

FT-IR spectroscopy has been shown to be an effective tool to classify bacteria, yeast, and fungi at the strain, species, and genus level. Compared to traditional microbiological techniques, IR sampling is less time-consuming. Microbiological applications for FT-IR analysis include a rapid method of identification and screening, quality control for food and pharmaceutical fields, and assessment of growth environment on cell and cell byproduct physiology.  

An IR plate-reader for biological samples is desirable for laboratories confronted with high-throughput demands, and the need to save resources through automation. This application note illustrates the use of an in-compartment XY plate reader to analyze yeast and discusses the resource savings involved through accessory automation.  

Yeast suspensions (30 µL) were pipetted onto a sample well of a 96-well reusable Si plate, and dried at 45 °C for 1 h for subsequent infrared transmission measurements. The 96-well Si plate was loaded into the PIKE Technologies XY Autosampler Accessory, an FT-IR sample compartment automated transmission/diffuse reflection accessory. The spectral range spanned from 4000 to 600 cm

 . Data collection time was 1 min. For transmission measurements the accessory's integrated DTGS detector is used to maximize the signal-to-noise ratio. Automated plate movement to each sample position is controlled by PIKE AutoPRO software, which also triggers data collection via the FT-IR spectrometer. 

Figure 1 shows a typical transmission spectrum of the dried yeast suspension. Some main spectral features include the C-H stretching vibrations from fatty acids between 3100–2800 cm

 , the amide I and amide II bands between 1800–1500 cm

 , and C-O-C and C-O-P stretching vibrations of polysaccharides between 1200–900 cm

 . A comprehensive list of absorption band assignments may be found in Naumann (1). Application of multivariate analysis allows differentiation between unique biological materials (2,3). 

Figure 1: Infrared transmission spectrum of dried yeast suspension.			

Through automation the number of IR samples analyzed per day may be greatly enhanced and potential sample and data entry errors decreased. Due to variability in biological materials, replicate sample measurements are often required. If four replicate measurements are made for one sample, 84 samples may be analyzed in a typical eight-hour work day. This equates to over 330 individual spectra collected without end user input, except to load the 96-well plates once the process is started by the AutoPRO software. 

In addition to biological applications, an automated XY plate reader may be used for polymer solutions and solvent residuals analysis. 

Infrared transmission spectra of microbiological suspensions may be collected by drying the suspension on an IR transparent plate. The use of an in-compartment XY plate reader accessory provides means for automated measurements via transmission or reflectance to increase laboratory capacity. 

				6125 Cottonwood Drive, Madison, WI 53719

				tel. (608) 274-2721, fax (608) 274-0103 

Yeast suspensions were dried and analyzed via FT-IR using an automated transmission sampling accessory. Up to six-fold sampling capacity increase may be realized through automation.

Author | PIKE Technologies, Inc.

Articles