Wisdom From Albert

1. Wisdom From Albert • "The important thing is not to stop questioning. Curiosity has its own reason for existing." • "If A is a success in life, then A equals x plus y plus z. Work is x; y is play; and z is keeping your mouth shut."

2. Proteins have a Hierarchy of structure Please describe that hierarchy and how each hierarchy fits into the next.

3. Bits of Chapter 5 • Hydrodynamics Covered in Biophysical • CD spectra • Circularly polarized light interacts with Chiral molecules • Helices and Beta sheets are Chiral even if they are made out of glycine. • Why • helices more chiral than beta sheets • why?

4. Bits of Chapter 5 • Fiberous proteins • historically important • First structures elucidated by x-rays • Gave us terms Apha helix Beta sheet • should review • Take home Quiz/homwork 6. Based on their molecular structures why can you wash silk in hot water and have to wash wool in cold?

5. Protein structure How do we look at proteins

6. How do we determine the structure of proteins • X ray Crystallography • NMR • Neutron diffraction • Electron diffraction.

7. X-ray Crystallography • About the Unit Cell • Why are X-rays used ? • Why X-ray diffraction ? • Growing Crystals • X-ray diffraction

8. About the Unit Cell • Crystals are three dimensional ordered structures than can be described as a repetition of identical unit cells. • The unit cell is made up of the smallest possible volume that when repeated, is representative of the entire crystal. • This unit contains an integer number of protein molecules • The dimensions of a unit cell can be described with 3 edge lengths (a,b,c) and 3 angles (alpha, beta, gamma). • The 3D location of atoms within a unit cell can be listed as their x, y, z Cartesian Coordinates. • Space groups describe the symmetry of a unit cell, of which there are 230 variations

10. Why are X-rays used ? Resolution is limited to ½ λAtoms have dimensions in Å Visible light has wavelengths of 100s of nanometers X-Rays have wavelengths of Å

11. Why X-ray diffraction ? • No known way to focus x-rays with a lens. • Diffraction patterns can be interpreted mathematically. • We use computers as a virtual lens, so on a monitor we can look at the structure of a molecule. • The x-ray diffraction from one unit cell would not be significant. Crystals are important because by definition they have a repeated unit cell within them. • The repetition of unit cells within a crystal amplifies the diffraction enough to give results that computers can turn into a picture

12. Growing Crystals • Mostly a trial and error process • Closely related proteins may require different conditions to crystallize • Some consistent principles • Protein must be relatively pure • Requires some sort of small ion salt • Precipitant usually involved. • Some way of gradually increasing the ppt in the system. • pH is usually close to the pI of the protein

13. Growing Crystals • More general principles • High concentration of protein • Low temperature tends to favor crystallization • Partial factorial screens now common • Dumb Luck • Numerous cases of finding conditions by accident during purifications on proteins

14. Methods of Crystal growth • There are various methods of growing protein crystals: • Vapor Diffusion -(Hanging Drop Method)This is probably the most common ways of crystal growth. A drop of protein solution is suspended over a reservoir containing buffer and precipitant. Water diffuses from the drop to the solution leaving the drop with optimal crystal growth conditions. • Batch crystallizationA saturated protein solution left in a sealed container to let the crystals grow. • Micro batch crystallizationA drop of protein solution is put in inert oil and left to grow. Here there probably is some diffusion of proteins into the oil, lowering the saturation over time.

15. Methods of Crystal growth • There are various methods of growing protein crystals: • Vapor Diffusion -(Hanging Drop Method) A drop of protein solution is suspended over a reservoir containing buffer and precipitant. Water diffuses from the drop to the solution leaving the drop with optimal crystal growth conditions. • Batch crystallizationA saturated protein solution left in a sealed container to let the crystals grow. (Almost always the way dumb luck works) • Micro-batch crystallizationA drop of protein solution is put in inert oil and left to grow. Here there probably is some diffusion of proteins into the oil, lowering the saturation over time.

16. Methods of Crystal growth • Free interface diffusionA container has levels of varying saturation. Microcrystals/ppt form initially in the highly saturated part, but as the solution mixes, it eventually only supports crystal growth. • Dialysis Similar to the previous, but with a semi-permeable membrane separating the layers. • Macro-seeding A crystal is grown in a highly saturated solution and placed in a less saturated one where only growth of the crystal will occur. • Micro-seedingA few crystals are grown, then crushed, and put into a final solution that combines them into a few nice crystals. This involves quite a bit of experimentation with solutions' concentrations to get the desired number of crystals.

17. X-ray diffraction • Single Wavelength diffraction • 1.542 Å most commen Resolution 0.771 Å • Multiple exposures required • Heavy atom derivitives required. • Data is relatively easy to interprete. • Ability to tell if the Data is worth anything • Equipment fits in a regular lab

18. X-ray diffraction • Laue Diffraction • Multiple wavelengths used • Requires a cyclotron to generate X-rays • Data is much more complex • Single good exposure often results in a structure • Able to use poorer quality crystals • Phase may be determined by over-tones • Able to image transient structures.

19. X-ray diffraction • Reflections occur in reciprocal space • Reflections follow Braggs Law • 2d sinΘn = nλ Symmetry elements critical to solving the structure.