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Protein Crystallography of Lysozyme
- 1. Protein Crystallography of Lysozyme Anil Chaturvedi, Ashley Davalos, Carlos Hernandez, Guillermo Llamas, Mabel Wong Department of Chemistry and Biochemistry, University of California Santa Barbara Santa Barbara, CA 93106 Introduction Lysozyme is a catalytic enzyme that is known to hydrolyze the 硫-(1 4) glycosidic linkages from the N-acetylmuramic (NAM) to N- acetylglucosamine (NAG) in the cell wall peptidoglycans of bacterial cell walls. Crystallography and x-ray diffraction are valuable techniques for determining the structure of proteins such as lysozyme. They are capable of revealing the geometry of substrate binding, and active site residues that may be crucial to the catalytic mechanism. Viable x-ray diffraction data requires large singular crystals that are often difficult to obtain. Successful crystallization is a trivial process that requires experimentation with many different precipitating conditions. The conditions can vary by precipitating agents, temperature, purity, pH, and concentrations. From the x-ray diffraction pattern the unit cell dimensions, angles and symmetry can be measured by using the program HKL view. The reflection data collected can be converted into an electron density map through the use of Fourier summations and other associated calculations. Amino acids can be fitted into the electron density map and this can be used for conformational analysis of peptides and proteins using Coot. Methods Results 油 Crystallization Diffraction 油 Electron Density 油 Conclusion References 油 Acknowledgements We would like to acknowledge Dr. Kalju Kahn for the guidance, valuable discussions, as well as the use of the Bioanalytical Lab and the X-Ray Analytical Facility in the Chemistry and Biochemistry Department at UCSB. We would also like to extend our gratitude to Kota Kaneshige and Istvan Szabo for providing extra guidance during this project. Precipitating agent Y2 Y1 X2 X1 Concentration 2 mg/mL Nothing Nothing Nothing Nothing 10 mg/mL Big shards Big shards Small hexagonal cubic Medium hexagonal cubic 15 mg/mL Medium shards Medium shards Medium hexagonal cubic Biggest hexagonal cubic 30 mg/mL Small shards Few small shards Big hexagonal cubic Medium hexagonal cubic 60 mg/mL Micro- crystals: Shards Few small shards Micro- crystals Micro- crystals 100 mg/mL Micro- crystals: Shards Micro- crystals: Shards Micro- crystals Micro- crystals Axis Point-to-point distance (a, b, c) h-axis a = 129.8 悩 k-axis b = 129.8 悩 l-axis c = 91.4 悩 Figure 1: The image on the left displays the result of the most successful crystal growth condition using precipitating agent X1 and buffer solution X. Table 1: Relates the trends juxtaposing the varying concentrations of lysozyme and precipitant agents. PEG 8000 produced the best crystals. 油 Precipitants Y1 and Y2 produced shards. 油 Lysozyme concentrations between 15 mg/mL and 30 mg/mL produced best results. By the hanging drop method the most successful crystals formed at a protein concentration 15 mg/mL, pH 4.5, 30% (w/v) PEG 8000, 1 M NaCl, 50 mM sodium acetate. 油 Analysis of the lysozyme diffraction pattern by HKL view revealed the unit cell dimensions to be a= 129.8 , b= 129.8 , and c= 91.4 , and that the crystals are tetragonal by these crystallization conditions. 油 The same program was used to determine the space group as P43212 based on the symmetry of the diffraction pattern. 油 The program Coot was used to fit amino acids into the electron density map of an 留-helical segment of a protein and elucidate its structure. This process can also be done for lysozyme but involves difficult calculations. 油 These methods outline a basic protocol for protein crystallography, but there are other considerations and techniques that better elucidate crystal structures. The three-dimensional structures of proteins are useful for structure-based drug design. Poor Best Table 2: The unit cell dimensions tabulated on the left were determined using the HKL View program. All the angles were 90 . Since a = b c, the unit cell is tetragonal. Figure 2: The three diffraction patterns displayed as pseudo-precession-stills of zones of reciprocal space show the symmetries with respect to h, k, and l axes. 油 From our analysis, the lysozyme unit cell is characterized by a tetragonal lattice system with variations in either a primitive or body centering and is defined by the following space group: P43212. 油 The space group is characterized by the apparent symmetries in the reflection data with respect to each axes caused by the rotation and translation of the unit cell. 油 Unit cells are specific to crystallization conditions. Lysozyme is known to form orthorhombic crystals with space group P21212, monoclinic crystals with space group P21, and tetragonal crystals with space group P43212. 油 The point group, which is the first number in the space group, indicates the rotational symmetry perpetuated by the crystal. 油 Figure 3: The three-dimensional positions of the segment YSVLFDMARE was fitted using three auto-fitting algorithms in Coot. 油 Real Space refine Zone: optimizes fit of electron density and preserves stereochemistry 油 Regularize Zone: optimizes stereochemistry 油 Rigid Body Fit Zone: optimizes the fit of the model to electron density Figure 4: Electron density map for the given peptide sequence. 油 An electron density map is generated by performing Fourier series of the intensities of the reflection data. Composition of the precipitating solutions X1 30% (w/v) PEG 8000, 1 M NaCl, 50 mM Sodium acetate, pH 4.5 X2 37% (w/v) PEG 8000, 1 M NaCl, 50 mM Sodium acetate, pH 4.5 Y1 8% NaCl, 100 mM Sodium acetate, pH 4.8 Y2 2% (w/v) MgCl2, 8% NaCl, 100 mM Sodium acetate, pH 4.8 1. Bhat, R. Timasheff, S. N. Protein Science. (1992); 1: 1133-1143. 2. Yao, Y et al. CrystEngComm. (2008); 10: 166-9. 3. Hu, Z. W. et al. Biological Crystallography. (2001); 57: 840-846. 1. Crystallization 油Lysozyme was crystallized using hanging drop technique under four different crystallization conditions on a Linbro plate. 油 Precipitating agent X1, X2, Y1, Y2 and buffer solutions X and Y 油 Six different concentrations of lysozyme: 2 mg/mL, 10 mg/mL, 15 mg/mL, 30 mg/mL, 60 mg/mL, and 100 mg/mL 2. Diffraction Unit cell dimensions, space group, and point group were determined using Measure in HKL View. 油 3. Electron Density 油Three auto-fitting algorithms in Coot were used to generate different positions of each amino acid. 油 Residues that visually fit the electron density map were verified using Density Fit Analysis tool.