Structural And Thermal Evaluation Of Bovine Serum Albumin Using CIRCULAR

DICHROISM (CD) Spectroscopy

Biomätteknik Linköpings Universitet

2013 Handledare: Madhan Anandapadamanaban

madan@ifm.liu.se

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1. Introduction The goal of this laboration is to understand the importance of circular dichroism (CD) spectroscopy in protein research. We will apply CD to study

1. secondary structures and 2. thermal stability

of the protein BSA (Bovine Serum Albumin). In the first experiment we will investigate spectra of bovine serum albumin in the wavelength range between 180nm and 260nm (far-UV-CD). The spectra recorded at far-UV-CD will be used for calculation of the secondary structure content of the protein. For this calculation CD programs such as Selcon3, CONTIN and CDSSTR will be used. Additionally, we will compare the acquired data with the already determined crystal structure of BSA (PDB: 3V03), to see how reliable our spectra and calculations are in terms of secondary structure content. The second experiment involves thermal unfolding of the protein at a fixed wavelength chosen by the student based on the full-scan spectra from the first experiment. A preliminary melting midpoint (Tm) will be calculated from experimental data after analysis in Excel.

2. Theory

2.1 Circular Dichroism Spectroscopy Circular dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as α-helix and β-sheet. The thermal unfolding of secondary structures as judged by the thermal melting midpoint (Tm) can also be analyzed in this region. The unfolding process gives information about the stability of the protein and can be related to other proteins and/or mutated forms based on the slope of temperature/CD-intensity-plot and the thermal melting midpoint (Tm) calculated from the graph. In practice, the CD instrument (spectropolarimeter will display the dichroism at a given wavelength of radiation expressed as either the difference in absorbance of the two components (ΔA = AL − AR) or as the ellipticity in degrees (θ) (θ = tan−1(b/a)), where b and a describe the rotation of the polarized light). There is a simple numerical relationship between ΔA and θ in degrees, i.e. θ = 32.98·ΔA. A CD spectrum is obtained, when the dichroism is measured as a function of wavelength. If two or more identical chromophores are in close proximity, the interactions between them can give rise to a sigmoidal CD spectrum. A summary of the most common peptide bond signals in CD spectra are shown in fig. 1. The helical signal dominates the spectrum if β-sheet and α-helix is present and a strong negative absorption with maxima at 208nm and 222nm and a positive absorption with maxima at 192 nm. The β-sheet signal shows instead a negative absorption at 215nm and a positive absorption at 198nm. A random coil formation results in an intense negative band at 198 nm.

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Figure 1: Three representative CD signals. The red line resembles an α- helical signal, blue a β-sheet and purple a random coil formation.

There are several methods in use to calculate secondary structure content from CD data. Basically, all of these methods assume that the spectrum of a protein can be represented by a linear combination of the spectra of the secondary structural elements, plus a noise term which includes the contribution of aromatic chromophores given in equation (1): θλ = ΣFiSλi + noise (1) where θλ is the CD of the protein as a function of wavelength, Fi is the fraction of each secondary structure, i, and Sλi is the ellipticity at each wavelength of each ith secondary structural element. In constrained fits the sum of all the fractional weights, ΣFi, must be equal to 1. The major methods of extracting structural information from CD spectra are multilinear regression, singular value decomposition, ridge regression, convex constraint analysis, neural network analysis and the self-consistent method.

2.2 Evaluation of CD spectra There are several programs available, calculating the secondary structure content out of CD spectra. They use different algorithms but are all based on protein sets as database. The structure and thus the secondary structure content of these proteins are already determined and are used as a reference. Programs available are CONTIN (ridge regression), SELCON3 (single value decomposition), CDSSTR. The programs need different input data. Usually one can say, the more wavelength range covered the more reliable is the calculation. Most of the programs need at least a range of 190-240 nm. It is absolutely necessary for these programs, that the concentration of the sample protein is as accurately determined as possible. Otherwise the data is even less reliable and the error is too big to use for publications or similar. Furthermore, the spectra should have a good signal-to-noise ratio. The thermal unfolding data will be analysed in excel or similar program to generate a graphic view which will be used for estimation of the thermal melting midpoint Tm.

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3. Experimentals Experiment 1: Structural determination (secondary structure) of BSA (this part of the lab will be performed in groups of two to three students at a time) In the wavelength range 190-260 nm we will record a spectrum for BSA using buffer-only as a blank. We will subtract this blank spectrum from the protein spectrum to obtain the true protein spectrum. The acquired spectra are recorded in Delta A units (delta-absorption instead of ellipticity). To proceed with the analysis, it is necessary to convert the data into the appropriate and required units. The program we will use in this course for determining the secondary structure content will be shown during the course. The name of the web-based program is Dichroweb and can be accessed via: http://dichroweb.cryst.bbk.ac.uk/html/process.shtml For each sample three scans will be recorded and the average converted into an ASCII-file, which is transferred to another computer, where the evaluation using Dichroweb will be performed. The result of the calculated secondary structure content will be compared with a crystal structure of BSA, which can be retrieved from the PDB-data base. http://www.pdb.org/pdb/home/home.do Experiment2. Thermal stability evaluation of BSA (this exercise will be performed jointly by the whole group due to time constraints). The unfolding of the secondary structure will be followed by measuring the Delta Absorbance of the protein at different temperatures (10°C-90°C, 5°C step) at a fixed wavelength. The data will later be analysed using an excel-based graph to identify a melting midpoint, Tm.

4. Lab Report The lab report should comprise the following:

1. Introduction. Why is this lab important? 2. Short background about CD-spectroscopy and the application to protein structure and

stability. 3. Experimental procedure (short) for both experiments. 4. Evaluation data of secondary structure content and comparison with crystal structure

data. 5. Evaluation of thermal unfolding of Bovine Serum Albumin (BSA). Graph and

estimated Tm. 6. Discussion and conclusion around results and source(s) of error.