ASSESSMENT OF PROTEIN SECONDARY STRUCTURE STABILITY BY FOURIER TRANSFORM INFRARED SPECTROSCOPY

Abstract

This study was designed to explore the potential for utilizing FTIR spectroscopy to predict the secondary structure of recombinant proteins and to follow the changes in protein secondary structure when it is exposed to increases in temperature. FTIR analysis has been compared and contrasted to both X-ray crystallography and CD. Frequently, FTIR predictions have correlated to the secondary structures defined by X-ray crystallography. There have also been comparisons made between FTER and CD which have indicated that both techniques are effective in predicting protein secondary structure. The stability of a protein is largely represented by its three- dimensional structure and is important when determining a proteins function, activity, and stability. Many proteins have a low conformational stability and are easily denatured by disrupting the weak bonding patterns that maintain the proteins structure. Other proteins may exhibit high conformational stability and can endure moderate to extreme conditions. The ability to predict protein secondary structure is important in determining the overall stability of the protein. In addition to X-ray crystallography and circular dichroism, Fourier Transfrom Infrared (FTIR) spectroscopy is used to determine protein secondary structure. The region of 1700-1500 cm'-1' contains valuable information concerning protein secondary structure. The type of secondary structure present in the sample can be determined and any changes in the structure can be followed. Amide vibrations, emanating from the amide bonds present in polypeptides and proteins, can be detected in this region. The amide I and amide II absorption bands are the most useful and commonly noted bands studied. The amide I band results from the C=0 stretching vibrations of the amide groups. Also adding to the band are the associated N-H bending and C-N stretching vibrations. The amide El band occurs from the N-1-I bending vibration of the amide bond. FTIR analysis is frequently limited by the interference of absorption bands associated with water over much of the fundamental region of the infrared spectrum including the region 1700-1500 cm'-1'. The interference from the water bands can be avoided by the adherence of the protein onto a film or by preparing a KBr disk. However, this can often limit the analysis of the protein in its native liquid state. Another technique described and employed in this study is the use of deuterium oxide (D20) in place of the water. There is no strong absorption from D20 in the region of interest. Advances in infrared spectroscopy have brought the implementation of several new techniques that can be used to analyze a substance. For liquid samples, a sealed cell consisting of two salt crystals encompassing the sample have been employed for many years. A newer technique for analyzing liquids requires the use of a zinc selenide crystal resulting in the method of attenuated total reflection (AIR). Extensive data from this study has allowed the comparison of the use of the sealed cell and the ATR. The ATR technique was demonstrated to yield accurate and consistent results without interference from external interactions, most notably the interference of water vapor in the atmosphere surrounding the crystal. The use of dry gas to purge the FT1R instrument and the sample compartment was utilized in this study and was shown to effectively remove water vapor from the atmosphere.