Laboratory Experiment pubs.acs.org/jchemeduc
Spectroscopic Characterization of Amyloid Fibril Formation by Lysozyme Jeffrey K. Myers* Department of Chemistry, Davidson College, Davidson, North Carolina 28035, United States S Supporting Information *
ABSTRACT: Hen egg white lysozyme is used as a model protein to study amyloid fibril formation (fibrillation). This three-week laboratory experience has been successfully implemented in an upper-level biochemistry course to introduce students to issues of protein conformation, spectroscopic characterization of conformational changes, and misfolding of proteins related to disease. The fluorescence and absorbance of amyloid-specific dyes are used to detect fibrillation; accompanying changes in secondary structure are revealed by circular dichroism. Potential small molecule fibrillation inhibitors are tested, and kinetics experiments are carried out to probe the effect of seeding with preformed fibrils on the rate of fibrillation. KEYWORDS: Upper-Division Undergraduate, Biochemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Inquiry-Based/Discovery Learning, Biophysical Chemistry, Conformational Analysis, Enzymes, Proteins/Peptides, Spectroscopy homologous to human lysozyme, whose fibrillation is related to systemic non-neuropathic amyloidosis.10 In this paper, a three-week (representing three lab periods) series of experiments is described that examines the fibrillation of lysozyme. The focus of the experiments is on spectroscopic methods for detecting fibrillation. Two amyloid-binding dyes (Congo red13 and thioflavin T14) are used to detect amyloid fibrils with fluorescence and visible absorbance spectroscopies, taking advantage of changes in the spectroscopic properties of the dyes upon binding. Additionally, circular dichroism (CD) is used to probe changes in secondary structure that accompany fibrillation. CD is a time-tested method for examining the structure of biological macromolecules; CD in the far-UV region of the spectrum is sensitive to protein secondary structure because of the differential absorbance of peptide bonds. Investigating the native protein structure with CD can provide an excellent introduction to protein structure in undergraduate laboratories15,16 and is the most convenient method for giving students hands-on experience in structure determination, although instrumentation is fairly expensive (but not in comparison to high field NMR or X-ray diffraction equipment) and the data are low resolution. Mechanistically, fibrillation is thought to occur via nucleation-dependent polymerization, with the initial nucleation process being the slow step.2 Slow nucleation leads to a significant lag phase before fibrils can be detected. Students explore this mechanism by testing the effect of seeding on the kinetics of fibrillation. Because inhibition of fibrillation is currently of great pharmacological interest, students also test simple organic compounds for their ability to inhibit lysozyme fibrillation.
B
iological function is intimately connected to the conformation of macromolecules, such as proteins. Many diseases are related to the adoption of “incorrect” conformations by proteins,1 and exploiting connections to human disease is a powerful way to relate biochemistry lab experiences to realworld problems. Despite these facts, undergraduate biochemistry laboratory experiments involving protein structure are uncommon. The goals for this series of experiments are to introduce students to the concept of spectroscopic characterization of protein structure, to help them understand the influence of environmental conditions and small molecules on protein conformation, and to expose them to fundamental characteristics of amyloid fibril formation (fibrillation). To achieve these goals, students experimentally monitor fibrillation of a protein, test potential inhibitors of fibrillation, and follow the kinetics of fibrillation with an eye toward understanding the mechanism of fibrillation. Studies that describe procedures for studying amyloid formation in an undergraduate teaching laboratory appear to be nonexistant. Amyloid fibrils result from protein misfolding and are large, ordered, elongated aggregates containing multiple polypeptides.1−3 Fibrillation is typically irreversible and is thought to begin from a partially folded conformation. Fibrils feature a large proportion of β-sheet structure organized in a stacked fashion where adjacent strands are held together via hydrogen bonding and adjacent sheets by side chain interactions. Amyloid has been recognized as a critical part in the pathogenesis of many human diseases, including Alzheimer’s and prion disease.1 Hen lysozyme is a small (129 amino acids), well-behaved enzyme that has proven useful in undergraduate biochemistry laboratories.4−6 Hen lysozyme has been found to fibrillate in a variety of conditions in vitro;7−12 the enzyme is highly © 2014 American Chemical Society and Division of Chemical Education, Inc.
Published: April 4, 2014 730
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EXPERIMENTS The following experiments are designed to be undertaken in three separate lab periods spread over three weeks and may be done individually or in pairs. The first two lab periods require around 3 hours of time, including pre lab discussion. The third lab period requires longer in order to perform the kinetics experiment (data collected once per hour for 6 hours or so). In week 1, students prepare two fibrillation samples of hen lysozyme in pH 2 buffer: sample 1 as a control and sample 2 where fibrillation is expected. Sample 1 is kept at 4 °C for a week and sample 2 is placed at 65 °C for a week. Students obtain fluorescence emission spectra of thioflavin T in phosphate buffer and with an aliquot of sample 1 (representing native, nonfibrillated lysozyme). Students obtain absorbance spectra of Congo red in phosphate buffer and with an aliquot of sample 1. During week 2, students acquire fluorescence emission spectra of thioflavin T in the presence of aliquots of samples 1 and 2 and absorbance spectra of Congo red in the presence of aliquots of samples 1 and 2. Students collect a far-UV circular dichroism spectrum of sample 2 (fibrillated lysozyme) to identify changes in secondary structure in comparison with a previously collected spectrum of the native protein. Students also prepare new fibrillation samples in pH 2 buffer (samples 3 and 4) to test inhibitors (sucrose and N-methylacetamide) and incubate those samples at 65 °C for a week. During week 3, students acquire fluorescence emission spectra of thioflavin T and Congo red absorbance spectra in the presence of aliquots of samples 3 and 4. Students run kinetics experiments on two fresh fibrillation samples in pH 2 buffer (samples 5 and 6) to test the effect of seeding. Sample 6 is seeded with an aliquot of sample 2. Both samples 5 and 6 are incubated at 65 °C for approximately 6 h. Aliquots are removed every 60 min and mixed with thioflavin T; a fluorescence emission spectrum of thioflavin T is acquired for each sample. Students analyze the data and prepare and submit a report. Detailed experimental procedures may be found in Supporting Information.
Figure 1. Far-UV circular dichroism spectra of native and fibrillated lysozyme, showing a dramatic change in secondary structure upon fibrillation. These spectra were deconvoluted with DichroWeb to give the secondary structure estimates given in Table 1.
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Figure 2. Lysozyme fibrillation detected by Congo red absorbance. Fibrillation sample 1 was placed at 4 °C for 1 week as a control; sample 2 was placed at 65 °C for 1 week. Note the distinct shoulder present in the spectrum for sample 2 near 550 nm.
HAZARDS The fibrillation buffer is acidic, so skin contact and ingestion should be avoided. Thioflavin T and Congo red dyes are not known carcinogens; however Congo red has mutagenic effects and is a suspected teratogen, so care should be used in dye handling and protective clothing and eye protection should be worn. Similar precautions should be used when handling Nmethylacetamide.
DichroWeb requires preregistration. Table 1 shows the percentages of different secondary structure types as given by analysis of the CD spectra in Figure 1 using DichroWeb and also shows the percentages from the crystal structure for comparison. CD usually gives secondary structure distributions comparable to the crystal structures of proteins, although for lysozyme, there was some disagreementnot uncommon in proteins with a high content of β-strands and β-turns.20 A marked increase in β-strand content and a marked decrease in the content of α helices were observed for the fibrillated sample, as expected. A slight increase in the content of disordered structures was also observed. Results from using Congo red absorbance as a probe are shown in Figure 2, including visible absorbance spectra of Congo red as the free dye and fibrillation samples 1 (control) and 2 (fibrillated). A red shift in the spectrum (or broadening, with increasing shoulder evident at around 550 nm) was taken as a positive indication of fibrils. Some red shift was observed with the control sample, indicating the dye might have some
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RESULTS AND DISCUSSION This lab module has been tested over three semesters (a total of 48 students) in an upper-level biochemistry class. Typical student-collected data are shown in Figures 1−4. Figure 1 shows the circular dichroism (CD) spectra of native lysozyme and fibrillation sample 2 after 1 week at 65 °C. Dramatic changes occurred in the spectrum as a result of fibrillation. Qualitatively, the shape of native lysozyme was that of a typical mixed α/β protein, whereas the fibrillated spectrum appeared to be dominated by β-sheet structure. Quantitatively, the content of secondary structure in each conformation was obtained using deconvolution analysis17 with available computer algorithms. Two online programs, SOMCD18 and DichroWeb19 were used for analysis; both are free although 731
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inhibitors added (sample 3 with 1 M sucrose, sample 4 with 1 M N-methylacetamide; NMA). Neither the free dye nor native lysozyme (sample 1) demonstrated a significant level of fluorescence (they are overlapping near the x axis in Figure 3); however, a dramatic increase in emission intensity was observed when the dye bound to fibrils. In general, the thioflavin T assay was more robust than the Congo red assay,21 and the change in the spectrum was much more obvious. Thus, the thioflavin T assay was used for testing inhibitors and in kinetics experiments. Several compounds were tested for inhibition of fibrillation, and 1 M sucrose and 1 M N-methylacetamide were found to strongly inhibit fibril formation (Figure 3). Sucrose presumably inhibited fibrillation via stabilization of the native state, whereas N-methylacetamide may have mimicked the polypeptide backbone, competing for hydrogen bonding interactions with the peptide groups in lysozyme and preventing interaction with neighboring strands in the fibril. As an extension of the experiment, students may choose to examine other potential inhibitors. Compounds known to affect protein stability, such as stabilizing osmolytes, denaturants, or salting-in/salting-out salts, are logical choices. Additional compounds are already known to inhibit lysozyme fibrillation, such as indole derivatives.10,22 Kinetic experiments showed clearly the effects of seeding with preformed fibrils. The presence of preformed fibrils in a fibrillation sample provided a scaffold upon which further fibrillation can occur, speeding up the process. The lag period typically associated with fibrillation2 was largely eliminated in the seeded sample (sample 6 seeded with 50 μL of sample 2), consistent with the belief that nucleation of the fibrils is the rate-limiting step (Figure 4). Students compared the mechanism of fibrillation with that of crystallization, which was dealt with previously in lab via crystal growth experiments using lysozyme.
Figure 3. Lysozyme fibrillation detected by thioflavin T fluorescence, demonstrating the dramatic increase in intensity when the dye is bound to fibrils (sample 2) and the inhibitory effect of sucrose (fibrillation sample 3) and N-methylacetamide (NMA, fibrillation sample 4).
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CONCLUSIONS Lysozyme is a convenient model protein with which to study fibrillation, an important type of protein conformational change related to human disease. The pedagogical benefits of such experiments were exposure to important spectroscopic techniques in biochemical analysis and emphasis of the connection of molecular structure to biological function and disease pathology. The learning goals of the lab were assessed mainly via inspection of the resulting lab reports, with particular focus on the presentation and interpretation of data, conclusions drawn from the data, and responses to postlab questions (Supporting Information). By the end of the lab, students were able to demonstrate the use of spectroscopic information to detect protein conformational changes, understand the mechanism of fibrillation, and understand the effect of small organic molecules on fibrillation at a molecular level.
Figure 4. Kinetics of fibrillation monitored by thioflavin T fluorescence demonstrating the effect of seeding with preformed fibrils. Fibrillation samples 5 and 6 were prepared and treated the same, except that sample 6 contained 50 μL of sample 2 to seed with preformed fibrils. Note the dramatic effect of seeding in reducing the (normally days-long) lag phase.
Table 1. Secondary Structure Content of Lysozyme from Deconvolution of Experimental Circular Dichroism Data from Figure 1 compared with Analysis of the Crystal Structure Native Fibrillated Crystala a
Helix
Strand
Turn
Disordered
0.28 0.09 0.36
0.26 0.35 0.09
0.21 0.22 0.32
0.28 0.34 0.23
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Crystal structure data from Hennessey and Johnson.17
ASSOCIATED CONTENT
S Supporting Information *
affinity for folded lysozyme, but the shoulder was much more distinct in the fibrillated sample. Results from using thioflavin T fluorescence as a probe are shown in Figure 3, including spectra of thioflavin T as the free dye, the two initial fibrillation samples (sample 1, control and sample 2, fibrillated) and two fibrillation samples with potential
Notes for instructors containing additional references, pre/post lab questions and answers, a list of needed chemicals, detailed experimental procedures, and student handouts with detailed procedures and instructions for CD data analysis. This material is available via the Internet at http://pubs.acs.org. 732
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(20) Sreerama, N.; Woody, R. W. A self-consistent method for the analysis of protein secondary structure from circular dichroism. Anal. Biochem. 1993, 209, 32−44. (21) Khurana, R.; Uversky, V. N.; Nielsen, L.; Fink, A. L. Is Congo red an amyloid-specific dye? J. Biol. Chem. 2001, 276, 22715−22721. (22) Morshedi, D.; Rezaei-Ghaleh, N.; Ebrahim-Habibi, A.; Ahmadian, S.; Nemat-Gorgani, M. Inhibition of amyloid fibrillation of lysozyme by indole derivatives - possible mechanism of action. FEBS J. 2007, 274, 6415−6425.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The author wishes to thank Felix Carroll and Ruth Beeston for helpful comments and suggestions, and all of those students who participated in lab classes where these experiments were developed.
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REFERENCES
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