Circular Dichroism of Globular Proteins
Name of Student
Institutional Affiliation
Experiment 2: Circular Dichroism of Globular Proteins
Results
b. Myoglobin Far UV
C Con A far UV
D Myoglobin near UV
E. Lysozyme near UV
F. Con A near UV
G. Week 2 Spectra 1
H. Week 2 Spectra 2
2. Qualitative comparison of CD spectra for Proteins with Different structures
Protein Minimum (nm) Minimum (nm) Maximum (nm)
Lysozyme 207.4 207.5 191.5
Myoglobin 222.2 222.1 193
Concanavalin A 224.1 195.7
3. For the lysozyme, the type of secondary structure that dominates is the – -structure because it has a double minimum at 207.4 and 207.5 and a strong maximum at 191.5. It is, therefore, a – -helical protein.
For the Myoglobin, the type of secondary structure that dominates is also the – – structure because it has a double minimum at 222.2 and 222.1 and a stronger maximum at 193. The type of secondary structure that dominates in this type of protein is the -helical structure of the protein.
For the Concanavalin A, the type of secondary structure that dominates is predominantly -Sheet protein because it has an intensity that is lower than the -helical proteins. It has a single minimum of 224.1. The type of secondary structure that dominates is the -structure of the protein.
4. The differences between the values of the recorded spectral minimum and the expected ones are small considering they fall in the range of the minimum for the different types of proteins. The differences are therefore minimum.
5. The far-UV spectra are within the range of values as those in the paper. It is very similar to the one in the paper. The process was therefore highly accurate and consistent to those of the actual data.
6. The near –UV spectra for lysozyme, myoglobin and concanavalin are similar in many forms. The three spectra have similar trends over the different wavelengths.It therefore means that they show similar properties in the near-UV region as evidenced by the shape of their overlay spectra. However, the difference comes in in the specific molar ellipticity values. They share similar trends.
7. From an analysis of the three-dimensional structures of the proteins at first glance, the number of Trp is 3 while the Tyr is 2. On the other hand, the Phe’s are 3 in number. The number of disulphide bonds, on the other hand, is 4.
8. Associated inhibition mechanisms in the buffered solution greatly cause the yielding of completely different results. The buffer solution greatly affects the absorbance of the protein complexes. This is because the amino acid sequence is altered or enhanced to an extent. Signals in the near UV are caused by the absorption of the aromatic amino acids. Changing the environment significantly affects the structure of the bonds. The spectra of the complexes are virtually different in the buffered solution because it affects the absorption spectrum.
9. The protein is unstructured and flexible in the solution form. On addition of the higher TFE, the protein becomes increasingly unstructured. It increases the flexibility of the protein and the formation of a helix-like complex. TFE is a structure inducing solvent. It, therefore, leads to the formation of a stable structure due to the effects of secondary stabilization.
Discussion
Proteins have secondary structures which can be determined by a range of methods. Circular dichroism spectra is a good method of determining the secondary structure of proteins. The Alpha-helical, beta-structural, beta-bends and other irregular regions in proteins are determined by experiment to show the different spectra. The three proteins were used extensively as the reference proteins using the near and far-UV characteristics. The experiment was effective at giving a pedagogical advantage since the methods that were used provided more details about the protein structures using the spectroscopic methods. The position of the maximum and minimum peaks is a good indication of the type of protein structure that is predominant in the protein complex. The amplification of the peaks shows that the UV-spectra obtained is concentration dependent. Coincidentally, lysozyme is a protein that is involved in many enzyme activities in the body like the natural defence against bacteria in the intestine of an organism. This interaction was confirmed in the far-UV spectrum as observed in the enzyme activity. Through accurate and flexible analysis, it is easier to determine the secondary structure of the proteins (Sreerama and woody, 2000).
Variation in the biological function of many proteins plays a significant role in medicine and sterilization. Organic solvents stabilize the secondary structure of proteins. Secondary transitions are easily observable in the far-UV of between 190 and 250mm. The concentration of the TFE causes a significant change in the conformation and structure of a protein. Noticeable changes in the secondary structure are observed consequently leading to a loss in the enzymatic activity. The results which are based on the molar Ellipticity are based on a mean molecular weight which is residual in degrees of wavelength (Greenfield, 2006).
Circular dichroism greatly depends on the secondary structure of proteins and polypeptides. CD is a light absorption spectroscopy type that effectively measures the absorbance of light. CD spectra that range between 260 and 180 are effective and can be analyzed by the different secondary structure types which are an alpha helix, parallel and anti-parallel. Dichroism is defined as the different absorption of radiation that has been polarized in different directions as a function of frequency or intensity. In the case of this experiment, it was circular dichroism because of the use of circularly polarized light. Ellipticity was used as the unit of circular dichroism (Johnson, 1999).
CD is used to measure the secondary structure of the proteins and polypeptides. However, the major disadvantage of the method is that it lacked a standard reference. CD effectively determines the secondary structure of protein elements like the alpha-helix and beta sheet. The CD spectra in the far-UV are used to predict the percentage of the secondary element in the protein structure.
References
Greenfield, N. J. (2006). Using circular dichroism spectra to estimate protein secondary structure. Nature protocols, 1(6), 2876.
Johnson, W. C. (1999). Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins: Structure, Function, and Bioinformatics, 35(3), 307-312.
Sreerama, N., & Woody, R. W. (2000). Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Analytical biochemistry, 287(2), 252-260.
