Lysozyme Lab

Part II: Advanced Techniques

In the upcoming weeks you will focus on advanced techniques in biochemistry.

  • Homology Modeling
  • Applications of Fluorecence
  • Applications of NMR
  • Isothermal Titration Calorimetry for studying Protein-Ligand interactions

Most techniques will continue the theme of working with enzyme Lysozyme. The labs will be strongly connected to the classroom topics (ie homology modeling, theory of fluorescence, protein stability and thermodynamics). Two of the labs will be connected in theme. You will use two techniques to monitor stability of lysozyme - fluorescence and NMR. So the in addition to your discussion of each result you should include some final analysis comparing the results generated from the experiments.

I am continuing to make updates to this page as we go so please ask if you find anything is missing or if you have questions.

Week 8-10: Denaturation of Lysozyme

Thermodynamic Analysis by Denaturation

Chemical and thermal denaturation can be used to analyze the kinetic and thermodynamic stability of a protein. Thermal denaturation is considered irreversible and gives kinetic parameters. Chemical denaturation, in contrast, is reversible, and gives thermodynamic parameters.

We will use two different techniques to monitor the two different denaturation experiments. Thermal denaturation will be monitored by NMR spectroscopy. Because NMR requires significant amounts of material, we will perform this experiment as a class, using commercial lysozyme.

Chemical denaturation will be monitored by intrinsic fluorescence. This is a much more sensitive technique, and can be performed on small amounts of material. Depending on the yield from your purifications, you may analyze your own lysozyme, pool material with other students, or use commercial lysozyme, if necessary.

Reading

Lehninger, pages 143-144

Hammes. Physical Chemistry for the Biological Sciences. Section 3.6, pages 57-60.


Chemical Denaturation of Lysozyme by Fluorescence

Overview

  1. Prepare GuHCl and enzyme in matching buffers
  2. Measure Refractive Index of stock GuHCl to determine concentration
  3. Use the scanning spectrometer to determine wavelengths of maximum emission for the native protein and the fully denatured protein
  4. Prepare blanks and samples in duplicate on a 96-well plate
  5. Read samples and blanks on Thermo Fluorescence plate reader
  6. Plot data to determine free energy of folding

Preparation of Solutions

Prepare 50 mL of a fresh stock solution of ~7-8 M guanidine hydrochloride in 0.10 M Tris buffer and adjust to pH 7.0. (Note: You cannot simply prepare buffer and then add GuHCl to it. Why?)

Determine the final concentrations of denaturant stock solutions by measurement of the refractive index[2]. Measure the refractive index (RI) of the buffer solution without GdHCl. Next, measure the RI of the stock GdHCl solution. The concentration of GdHCl can be calculated according to the equation:

            Molarity (M) = 57.147(ΔN) + 38.68(ΔN)2 – 91.60(ΔN)3                              (ref [3])

ΔN is the difference in refractive index between the GdHCl solution and buffer (or water). There are also several online calculators based on the above equation.

Prepare 50-100 mL of 0.10 M Tris and adjust the pH to 7.0.

Prepare ~100μM protein stock in 100mM Tris buffer at pH 7.0. Depending on the initial concentration of the enzyme this may be best accomplished by buffer exchange on a PD10 column. - A PD10 column is a small size exclusion column that allows for the separation of high molecular weight substances (like proteins) from low molecular weight substances (ie buffers and salts). When a mixture of large and small molecules are applied to the top of the column the large molecules will do not diffuse into the pores in the column matrix are eluted first, usually within the void volume. The void volume is the column volume outside the matrix. Small molecules diffuse in and out of the pores to varying extent. They have a larger accessible column volume than the large molecules and therefore they elute after the large molecules. -Equilibrate a PD10 column with your Tris buffer by washing with 5 column volumes of the buffer. Apply 100-500μL of a concentrated sample (~ 5-10 mM) of your protein to the top of the column and allow it to bind. Elute the protein with 6-mL of tris buffer collecting 1-mL fractions in microfuge tubes. Identify the fraction(s) containing your protein by UV-vis at 280 nm. Pool samples containing your protein and determine the concentration by Bradford analysis or by UV-vis. It may be necessary to concentrate the protein in a Centricon until the concentration is ~ 100μM.

Sample Preparation

A series of solutions containing a fixed amount of lysozyme (3 μM) and varying concentrations of guanidine hydrochloride (0 –7.0 M) should be prepared from the stock solution.

The final volume of each sample was should be 200 μL in the wells. Make sure the samples are well mixed by gently pipetting up and down using and 200μL pipet set to 100μL and without introducing air. Allow the solution to incubate for at least 1 h at RT or overnight at 4oC. The advantage of the plate reader is that it allows for fast sample processing under many conditions. This allows you to record several excitation wavelengths and emission wavelengths. Measure the fluorescence spectrum of each blank and sample at an excitation wavelength of 280 nm (if your protein contains Tyr and Phe you can remove contributions from those residues by using an excitation of 295nm), and the emission wavelengths of 320nm, 340nm, and 360nm. All experiments should be done at a controlled temperature (~25 °C).

Instructions for Instrumentation

Analysis of Data

The denaturation of lysozyme will be analyzed using a two-state model.

 

 

Equilibrium

Protein denaturation curves often to exhibit a sigmoid dependence of measured parameter on the denaturant concentration[4]. Your collected fluorescence data will likely fit to the sigmoidal equation:

HillEq. 1

Equation 1 is commonly called the Hill equation in which y is the measured parameter (Em intensity at 340nm, ratio of 360nm/330nm, peak area, peak maximum, etc.), y0 is the baseline value of the measured parameter at low denaturant concentration, a is the difference between the maximum value of the parameter when the protein is completely unfolded and the baseline value (ymax - y0), x is the denaturant concentration, b is the Hill coefficient necessary to fit the data, and c is an interaction constant, equal to the denaturant concentration where ΔG = 0.

Fit your data to equation 1 using KaleidaGraph. The important parameters to draw out are the baseline and maximum values of the measured parameter estimated from the fit. They will be used in the next step.

Using Excel estimate the fraction of protein in the unfolded state (fU) at every denaturant concentration, as shown in Equation 2. It may be necessary to leave off data collected at the very low and very high concentrations of denaturant.

                                                           Fraction UnfoldedEq. 2

Next create a column to calculate the apparent equilibrium constant for unfolding (KU) at each value of fu for the denaturation process using Equation 3. and the free energy

                                                           EquilibriumFuFn Eq. 3

From the values of KU you can estimate the free energy for unfolding at each concentration of denaturant using the expression:

                                                           FreeEnergyFu Eq. 4

What is the trend in free energy of unfolding as the denaturant concentration increases? Is this expected?

Finally, you can estimate the free energy of unfolding at zero concentration denaturant by extrapolation using equation 5.

                                   FreeEnergyUnfolding Eq. 5               

Plot ΔGoU[d] versus denaturant concentration [d] and fit the data to the linear relationship above. The y-intercept is an estimate of the free energy of unfolding in buffer. For additional ideas for discussion consult the Proteins Textbook by Whitford.



Kurtin and Lee. Biochemistry and Molecular Biology Education (2002) vol. 30 (4) pp. 244-247

[2] Protein Structure: A Practical Approach, 2nd Ed., Creighton, p306.

[3] Nozaki, Y. (1972) In Methods in enzymology, Vol 26, p 43, Academic Press, NY

[4] C. Pace in G. D. Fasman, Ed. (1975) CRC Critical Reviews in Biochemistry, Vol. 3, pp. 1–43, CRC Press, Cleveland, OH.


Denaturation by NMR

Reading

Review reading on NMR from week 8 and denaturation reading on previous page

Introduction

Hydrogen bonding significantly affects the electronic environment of amide and alpha protons in a protein backbone. This change in electron density can be monitored as a change in chemical shift by 1D or 2D NMR.

We will be using 1D 1H NMR to monitor the thermal denaturation of lysozyme. We will first record the NMR spectra of the sample at a variety of temperatures. We will identify peaks that change in intensity as the sample is heated, then integrate those peaks in each spectra. In week 12, we will fit those values to a melting curve in order to determine the denaturation parameters.

NMR Referencing

NMR determines the chemical shift of each proton relative to a standard (trimethyl silane, TMS, is common in organic solvents). In biomolecular NMR, a water-soluble standard is needed, and DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) is used instead. This standard will be defined as ‘0.000’ ppm. The incorporation of a standard will allow us to compare the chemical shifts of peaks across different NMR runs.

DSS

The NMR program will initially reference the spectra to D2O, but the chemical shifts of H2O and D2O vary significantly with temperature. Therefore, when you first process data collected at high temperature, it will appear as if the entire spectrum has shifted. Referencing all the spectra to DSS will fix this problem.

Kurutz, J. “Chemical Shift Referencing for Biomolecular NMR.”
http://cavanagh-lab.bch.ncsu.edu/new/intranet/data_collection/varian_referencing.pdf


Procedure

As a class, prepare three lysozyme samples for NMR. Determine the amount of lysozyme necessary to prepare 750 uL of ~5 mM lysozyme. Weigh this out and dissolve in 675 uL distilled H2O.

Make up 0.5 mL of 20 mM DSS dissolved in D2O (MW of DSS is 218.3 g/mol). You might need to make a more concentrated stock solution initially in order to weigh accurately.

Add 75 uL of the DSS/D2O to the lysozyme sample, so the final solution is 10% D2O + 10 uM DSS.

Spin the tube for 1 minute at full speed in the microcentrifuge to precipitate any fibers or undissolved lysozyme.

Transfer the supernatant to a clean NMR tube with a P200. (The P1000 tends to give bubbles.)

Small groups of students will take turns acquiring 1D 1H NMRs of the lysozyme at rising temperatures. Determine a reasonable number of temperatures for a first pass, bearing in mind that the instrument and sample take ~fifteen minutes to equilibrate between each sample.

After the first pass determines the approximate melting temperature, the second and third samples will be used to monitor the denaturation a second time with finer temperature steps.

NMR Procedure

Step by step NMR instructions

Analysis

Each spectrum will be phased and the chemical shift adjusted to the DSS standard. Once the spectra have been calibrated, they can be overlaid to determine several peaks that appear or disappear as the denaturation occurs.

As a class, integrate these peaks in each spectrum and plot them versus the temperature to determine the melting profile of lysozyme.

 

Analysis of Thermal Denaturation

Reading

Yu, C, et al. Folding of the SARS Coronavirus Spike Glycoprotein Immunological Fragment (SARS•S1B).” Biochemistry 2005, 44, 1453-1463. (Focus on the results sections as an example of the kind of analysis we are doing.)

Reread: Lehninger, pages 143-144

Hammes. Physical Chemistry for the Biological Sciences. Section 3.6, pages 57-60.

 

Analysis of NMR Data

The NMR data will likely fit to the sigmoidal equation:

daum equation Hill                          

Hill equation, Eq. 1

where y is the measured parameter (integration in this case), y0 is the minimum value of the measured parameter, a is the difference between the maximum value of the parameter and the baseline value (ymax - y0), x is the temperature, b is the Hill coefficient necessary to fit the data, and T is an interaction constant, equal to the temperature where ΔG = 0.

Fit your data to equation 1 using KaleidaGraph. Determine the denaturation temperature (Tm) and the max and min values.

For each data point, determine:

  • Temp recip
  • signal daum equation
  • recip alpha
  • ln alpha
  • Then plot
  • Linear heat denfor each data point and fit to a linear curve.
  • The slope should be –E/R, where E is the desired parameter (energy of denaturation), and R is the gas constant.

Analysis Questions:

  1. Did you have sufficient data points to accurately estimate y0 and ymax? Comment on the quality of the sigmoidal fit.
  2. How did you decide which data series to fit?
  3. What could you do differently to improve data collection or analysis?
  4. What was the TM of the sigmoidal fit? Does that fit reasonably well with your estimates for the TMs of the other data series that weren’t fit to the Hill equation?
  5. How does the Efolding for lysozyme compare to that of an average protein? to the literature value for lysozyme? (Include citations.)
  6. How does the folding for lysozyme (measured by thermal denaturation) compare to its Gfolding (measured by chemical denaturation)? Is this expected?
  7. Provide a final summary inegrated the results of both experiments

Week 10: Binding of ANS to BSA

 

Overview

A. Review the Process of Fluorescence
B. Effect of Solvent Polarity on ANS Fluorescence
C. Binding of ANS to BSA
D. Scatchard Plot and Calculation of Kd and n (number of ANS molecules/BSA)

Part A. Review the Process of Fluorescence

In this experiment we will probe hydrophobic binding sites on molecular surface of BSA using the fluorescent probe 1-Anilino-8-naphthalene sulfonate (ANS). ANS binds to hydrophobic pockets in enzymes. ANS is virtually nonfluorescent in aqueous solution but is highly fluorescent in nonpolar solvents. The binding of the probe to hydrophobic sites can be monitored by an increase in fluorescence intensity.

  • Review reading on fluorescence spectroscopy from class.
  • Research ANS and record a few common applications of this fluorescent probe.

 

Part B. Effect of Solvent Polarity on ANS Fluorescence

Prepare or obtain 1 mM ANS stock solution in ultrapure, filtered water. Prepare 4-6 solutions, 5 mL each, of varying concentration of ethanol in water. For example 0, 25, 50, 75, 90, 100%.

Record the emission spectrum of 
ANS (20 μM) in 100% ethanol using an excitation wavelength of 370nm. You will need to determine the optimum parameters for the instrument and emission range and the appropriate blank solution. Determine the wavelength of maximum emission.

Operating Instructions for BioTek Fluoresecnce plate reader

Repeat for the emission spectrum of ANS in the other solvent mixtures using the same parameters as determined in above.

Create an overlay of the various spectra to include in your notebook. Discuss the effects of solvent polarity on the emission spectrum of ANS. Does the quantum yield change? Wavelength of maximum emission?

Part C. Binding of ANS to BSA

Prepare or obtain 100 mM potassium phosphate buffer, pH 7.4.

You will measure the binding of ANS to BSA by monitoring the change fluorescence intensity as increasing amounts of ANS are added to a fixed concentration of BSA.

This can be done by one of two methods.

1) Direct titration of ANS into a solution of 10 uM BSA.

2) Prepare several tubes containing 10 uM BSA in buffer and increasing concentrations of ANS.

For procedure 2, prepare several tubes of 10 μM BSA in buffer and increasing concentrations of ANS ranging from 0 to 100 μM. It is important for the upcoming calculations that you have several points in the 0-10 μM range. Run a total of 12-15 samples in the complete range of ANS concentrations. Measure the fluorescence intensity using and excitation wavelength and emission wavelength determined previously. Plot fluorescence intensity (F) versus [ANS] in μM.

Part D. Scatchard Analysis.

We will follow the procedure shown in Möller and Denicola. Study of protein‐ligand binding by fluorescence. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION (2002) vol. 30 (5) pp. 309-312