I. Introduction
Advances in recombinant DNA technology and bioinformatics-based data analysis have driven the explosion of structural biology in the last two decades (Kelly, Jess, and Price 119). This has resulted to the identification of at least 2000 proteins annually and a growing need in performing structural studies in these new proteins. The current trend involves the concern of analyzing protein structures close to its natural conditions (and other conditions) and of measuring their rates of structural changes, an essential factor in their biological functions. The technique of circular dichroism (CD) has gained an increasing popularity in addressing these purposes. Variations in this techniques have also been noted such as electronic CD, vibrational CD, and fluorescence-detection CD among others.
CD represents the differential absorption of the two circularly polarized components of light, which are of equal magnitude, moving towards the opposite circular directions: clockwise and counter-clockwise (Kelly, Jess, and Price 119-120). Passing this light through a protein sample under study will be recombined to regenerate the optical radiation in the original plane if the polarized components are either not absorbed in the sample or absorbed equally. However, when differences in absorption occurs, an elliptical polarization occurs and is picked up in and observed through an optically active chromophore. Then, as a function of wavelength, the CD instruments, often referred to as ‘spectropolarimeters’, measure the absorbance difference, which the machine interprets mathematically and reports in an understandable graphical way.
The present laboratory experiment is significant in understanding better the mechanism of performing a CD measurement of a protein sample, a catalytic enzyme called lysozyme. It is a 14 kDa glycosidase enzyme containing of two subunits, one being predominantly α-helical and another being primarily β-sheet (Helmfors 7). It has four disulphide bonds, one of which links the two subunits. It is the first line of defense against bacterial infection due to its capability to cleave the glycosidic bond of the bacterial cell wall’s peptidoglycan layer (8). Lysozymal folding occurs in the oxidizing environment of the endoplasmic reticulum prior to Golgi apparatus secretion. Synthesized in hematopoietic cells, lysozymes can be found in leukocytes (e.g. granulocytes, monocytes, and macrophages) and bodily fluids (e.g. tears, saliva, blood, and breast milk).
Lysozymes, like other proteins, must be folded correctly to perform their intended functions (Helmfors 2). However, due to mutations, environmental factors, or simple complexity of the folding process, misfolding occurs naturally and is more common than rare. The human cell isolates and degrades misfolded lysozymes and further inhibits their formation. However, when the level of misfolded proteins is too large for the normal processes of the cell to cope, it will result to ailments (e.g. lysozyme amyloidosis) (6).
Moreover, the experiment will also enhance knowledge in spectropolarimetry and familiarity in the use of the spectropolarimeter as well as a better understanding of the theoretical and scientific background of such important processes as recrystallization, the melting process, re-freezing, and the CD concept itself as well as the polarization of light as a means to better understand the submicroscopic and essentially molecular structures of proteins, such as lysozymes.
This paper initially discusses initially describes in section II the mechanical aspects of the experimentation, such as the materials and equipment, followed by a simplified description of the sample preparation, which includes protein production and purification as well as sample storage, recrystallization, and the CD spectropolarimetry. Section III contains the presentation and analysis of results, while section IV provides the discussion.
II. Experimental
2.1 Materials
The primarily materials used include: Glass capillary, 1 piece; pure protein stock (as standard); lysozyme in mM sodium phosphate buffer (pH 7.5); and reagent blank.
2.2 Equipment
Metal block or oil bath, 1 piece (as the heat source for recrystallization)
Spectropolarimeter (Jasco J-810), 1 unit (as the measuring instrument)
2.3 Procedure
2.3.1 Protein Preparation
The protein samples were often prepared from over-expressed gene encoders found in suitable host systems (e.g. bacteria, lower eukaryotes, or insect cells) (Kelly, Jess, and Price 124). Protein purification was facilitated by adding a small gene tag (e.g. hexa-His tag) or by expressing the protein as a fusion protein with glutathione-S-transferase (GST) or maltose-binding protein (MBP). The tag may be removed, if interferes with protein folding or stability, using a protease. The GST or MBP should also be removed before structural studies are performed as it adds to spectroscopic signals.
The protein samples need to be at least 95% pure on SDS-PAGE using Coomassie Blue or silver stain and confirmed by mass spectrometry (Kelly, Jess, and Price 125). Nucleic acid or oligonucleotide fragment contaminants can be easily detected spectrometrically with an absorbance ratio of 0.6. Moreover, protective agents or buffer ions, which might cause problems with CD, must be removed by dialysis or gel permeation. Any insoluble contaminants will cause artefacts during measurements.
The folded state of proteins in solution are usually soluble and stable at a narrow pH range (Kelly, Jess, and Price 125). Protective agents (e.g. salts or osmolytes) imitates cellular conditions better in dilute buffer solutions. Glycerol (diluted to 50% v/v) also keeps proteins in solution at -20oC without freezing.
2.3.2 Recrystallization
Well-formed minerals with sharp edges indicates sharp melting points. Melting must be accomplished through a slow increase in temperature. The initial melting temperature (25oC) was noted as soon as the protein sample starts to melt and the maximum melting temperature (80oC) when the sample has fully melted. The melted sample often refreezes in the capillary tube with heat source removal. This refrozen solid is a large collection of highly refined crystals. Refreezing is accomplished in this study using ice and measured at the temperature of 50oC.
2.3.3 Circular Dichroism Spectropolarimetry
The concept of CD spectropolarimetry relies primarily on the excitation of electronic transitions in the amide group of lysozyme. Since lysozyme is an optically active macromolecule, it will exhibit differential absorption of circular polarized light through CD spectropolarimetry (Micsonai, et al. E3095).
After blanking the spectropolarimeter, the lysozyme sample was run in the spectropolarimeter at the far-ultraviolet (UV) spectral range of 190 nm to 260 nm) (cf. Micsonai, et al. E3095) and temperature ranges of increasing temperatures between 25oC and 80oC (as the temperature knob was switched up and recorded at 25oC, 40oC, 50oC, 60oC, 70oC, and 80oC) and then declining temperatures between 70oC and 50oC (with ice application and recorded at 70oC, 60oC, and 50oC). Printouts of absorbance and wavelengths are used to plot together lysozyme behavior at 25oC, 80oC, and 50oC.
III. Results
3.1 Circular Dichroism at Minimum Temperature (25oC)
The lysozyme sample at the initial and minimum temperature of 25oC showed an original negative CD detected between the 190 nm to 192 nm spectra, indicating at-rest right-rotating molecules at low energy levels (Fig. 3.1). Continued scanning at 0.5 seconds per scan (up to 8 scans) observed an increased activity in left-rotating lysozyme molecules as more heat built up, which is detected between 193 nm to 199 nm. It peaked to 11.7 Ao at 195 nm. Thereafter, the lysozyme molecules resumed its natural right-rotating structural behavior between 206 nm and 209 nm, which dipped to a maximum of -17.9 at 208 nm.
Prolonged heat exposure managed to slightly increase in left-rotating molecules between 211 nm to 236 nm and still failing to keep up with the predominant right-rotating lysozyme molecules, indicating structural resistance against change. Nevertheless, the lysozyme molecules managed to overcome the right-left rotational behavior impasse at 239 nm with 2.16 Ao but tapered off towards the 260 nm spectrum between 2.16 to 7.34 Ao, showing a prolonged almost flat absorbance curve almost halfway throughout the 70 nm UV range and apparently less denatured structurally on the basis of its continued capability to express counterclockwise optical behavior.
This optical behavior indicates an inherent right-rotational structure of this enzymatic protein, which can only be moderately changed at prolonged exposure to low heat level and detectable at higher ultraviolet spectra between 239 nm and 260 nm. Moreover, lysozomal structure apparently showed resistance against structural change at low temperature level. Furthermore, in this spectral range, the enzyme exhibits weak but broad transition, which Kelly, Jess, and Price (121) correctly predicted.
3.2 Circular Dichroism at Maximum Temperature (80oC)
At maximal temperature of 80oC, the lysozyme behaved differently from its exposure to the lowest temperature. Despite the high temperature exposure, the protein molecules failed to initially overcome its inherent right rotating structures, simply hovering between -1.82 Ao and -17.82 Ao from 190 nm to 237 nm (Fig. 3.1). Left rotating structures merely started overcoming its natural right rotating behavior from 243 nm to 260 nm. This behavior relatively mirrored the lysozymal structural behavior at low temperature condition, including the weak broad transition, which Kelly, Jess, and Price (121) mentioned. Nevertheless, this behavior is far more muted than its behavior in the low temperature condition. In fact, it barely surpassed the equilibrium level, running only between 0.13 Ao and 2.12 Ao.
Figure 3.1: Absorbance Readings of Protein Sample in Temperatures 25oC, 80oC, and 50oC
At 216 nm, the absorbance almost mimicked that in the minimum and initial temperature with a difference of only 1 Ao towards 232 nm, after which it simply settled around the equilibrium point but almost equidistant with the 50oC absorbance curve towards the end of the UV spectrum. Evidently, the high temperature at the beginning of the run and throughout the spectrum apparently and effectively suppressing lysozyme’s potential susceptibility against structural change, favoring the left-hand circular direction and preventing lysozyme from ever expressing an opposing optical behavior, that is, opposite to its natural right-side circular optical behavior. The lysozyme behavior under maximum heat exposure apparently either increased the structural resistance of the enzyme against heat-induced optical changes or the high heat level so denatured the protein upon quick-heating it became unreactive to further heat stimulation after 201 nm to exhibit an L optical behavior.
3.3 Circular Dichroism at Minimum Cooled Down Temperature (50oC)
The cooling down procedure measured at 50oC showed a contrasting behavior to the heating up conditions, particularly in the initial spectra between 190 nm and 194 nm. Overwhelming left-hand rotatory structures dominated the right-hand rotating optical structures at 97.17 Ao at the onset’s 190 nm of the last measurement temperature, declining to almost half (49.5 Ao) at 192 nm and further to more than five times (18.11 Ao) at 194 nm (Fig. 3.1). This behavior provides an interesting insight into the lysozyme interaction with heat and cold temperatures. Since the single lysozyme sample was exposed serially from the lowest but increasing temperature up to the maximum temperature and to the lowest cooled down temperature, the denaturation theory entertained in section 3.2 may not be irreversible at all as the results tended to be assumed so. Instead, the lysozyme apparently uncoiled or unfolded upon boiling and refolded during the refreezing step of the procedure as evidenced by the very high absorbance level without undergoing denaturation. Otherwise, such high absorbance levels could not have occurred. These transitions were observed in the 25oC exposed sample but only in a relatively smaller degree, which is around nine times smaller within the 190 nm to 199 nm even if the 25oC heated lysozyme left-side rotation delayed by around 4 nm. Nevertheless, the unfolded lysozyme protein structure showed responsiveness to declining temperature and began to fold back into its original place. Thereafter lysozyme never went back upwards beyond the equilibrium point from its lowest dip of -20.1 Ao at 208 nm. It did manage to move towards the equilibrium at 260 nm. However, this curved appeared to be closely, even almost equidistant relative to each other, paralleling the curves of the 25oC and 80oC temperature samples.
3.4 Overall Optical Lysozyme Behavior across Heat Levels
The parallel flat-like curve of the three lysozyme absorbance behaviors under different temperature levels indicates a consistency in optical behavior from 209 nm to the maximum UV range of 260 nm, indicating a predictable lysozyme behavior at this specific wavelength. This behavior reflects an enduring optical behavior, which Kelly, Jess, and Price (121) contended as an evidence of a common transition behavior.
Apparently, these optical behaviors at different thermal levels differ more significantly only in the initial transition phase between 190 nm and 203 nm wherein cooled down lysozyme exhibited a very high left-hand rotatory behavior almost reaching 100 Ao level while absorbance at 50oC managed to transition towards left-hand optical rotation from 192 nm to 200 nm, or a range of 8 nm, indicating less heat denatured lysozyme while as absorbance at 80oC apparently experienced denaturation in a quick melting condition, disabling it from expression optical rotation beyond the equilibrium in the higher UV range.
Moreover, it is apparent that high initial absorbance and high initial temperature may have somehow impacted on the ability of lysozyme to optically change (i.e. change the lysozyme molecular structure thereafter) halfway in the UV spectrum range. Given that, light absorption and heat exposure determines the optical behavior, which reflects the structural changes experienced in the lysozyme molecule. And, since the near UV spectrum behavior of lysozyme reflects the action of its peptide bonds, these peptide bonds are susceptible to both conditions, specifically as lysozyme and more generally as protein.
IV. Discussion
Plane light has two circular or rotating polarized components of equal magnitude moving towards two opposing directions: left-handed (L) or counterclockwise and right-handed (R) or clockwise (Kelly, Jess, and Price 119). When biological compounds, such as proteins, undergo structural changes, plane light passing through it will also undergo unbalanced polarized rotation, which measurement methods such as circular dichroism, can detect. In the current experiment, which used a catalytic protein called lysozyme, such optical imbalance had been detected.
In the lower ultraviolet spectrum used in the study (190 nm to 260 nm), Kelly, Jess, and Price (122) observed that the absorption observed from 190 nm to 240 nm is largely due to the activities of the peptide bond. This explained the relatively stronger optical behaviors of lysozyme in this UV spectral range in all three temperatures. This also explained the unfolding behavior of lysozyme in melting temperatures, which the buffered solution may have provided protection from significant heat-related protein denaturation. In effect, the near biological condition wherein the lysozyme was placed enabled it to respond almost like in biological settings; thus, refolding back effectively upon the removal of heat by icing. This information is important to understand the inhibitive impact of boiling heat to the function of proteins, particularly lysozyme, as a consequence of unfolding. Conversely, refreezing also showed reversible refolding when temperature is lowered gradually.
Kelly, Jess, and Price also observed an initial occurrence of transition centered around 220 nm, which is relatively weak and broad (121), observed optically as a flattened absorbance curve. However, the more intense structural transition apparently observed around 190 nm was expected.
The largely right rotatory behavior of lysozyme at low temperature level (25oC) and at absorptions detected below 240 nm indicate the increased activities of peptide bonds acting as chromophores (Kelly, Jess, and Price 121). The weak but broad absorption bands observed towards around 260nm indicates the presence of disulphide bonds. Kelly, Jess, and Price (121) expected weak but broad transition located around 220 nm and a more intense transition at 190 nm, which also occurred in the laboratory experiment.
In lysozyme, the transition, however, is largely right rotational in nature, an apparently characteristic structural behavior. Kelly, Jess, and Price (121) pointed out the presence of a-helical content in the lysozymal peptides at 208 nm and 222 nm.
The results in section III supported the contention that CD is an effective approach in understanding the structural behavior of protein (in this case, lysozyme) when subjected to changes in its environment (in this case, thermal environment). As such, the laboratory experiment showed the effectiveness of CD spectropolarimetry in studying macromolecules like proteins in medical laboratory exercises at school.
Moreover, the results also enriched the understanding of spectropolarimetry as a measuring tool in CD analysis. An important reason for the methodological effectiveness resides from the optical activity of lysozymes and other proteins (Micsonai, et al. E3095). However, its effective use in the analyses of micromolecules cannot be demonstrated in this experiment. Perhaps a future laboratory experiment can explore this possibility.
Furthermore, the comparative absorbance curves of lysozyme in various temperatures provides a lesson on the potentially destructive impact of high environmental heat levels to human biological components, such as proteins, a class of biological substances that includes such critical substances as enzymes (like lysozymes). These substances participate in the efficient functioning of the body due to its catalyst function wherein most bodily metabolic processes highly depend.
V. Works Cited
Helmfors, Linda. “Understanding the Dual Nature of Lysozyme: Part Villain – Part Hero: A
Drosophila melanogaster Model of Lysozyme Amyloidosis. Linkoping Studies in Science and Technology 2014, Dissertation No. 1574: 1-89. PDF file.
Kelly, Sharon M., Thomas J. Jess, and Nicholas C. Price. “How to Study Proteins by Circular
Dichroism.” Biochimica et Biophysica Act 2005, 1751(1): 119-139. PDF file.
Micsonai, Andras, Frank Wien, Linda Kernya, Young-Ho Lee, Yuji Goto, Matthieu
Refregiers, and Jozsef Kardos. “Accurate Secondary Structure Prediction and Fold Recognition for Circular Dichroism Spectroscopy.” Proceedings from the National Academy of Sciences 2 Jun. 2015: E3095-E3103. PDF file.
