Proteins Lecture Lecture 4 slides Assignment is posted on line apparently.

Need to know for a midterm : to identifying lattice motif, asymmetric unit and unit cell. Make sure that the asymmetric unit is truly asymmetric. ( example: a heart shape is symmetric, so the asymmetric unit is only half a heart. ) Slide 28 (repeated exactly what was on the slide) Slide 29 ( explanation in slide 30) Slide 30 - Concentration of the hanging drop (containing protein) is lower than the precipitant in the beaker. Water will move according to concentration gradient to concentration the hanging drop containing the protein of interest. Because vapour diffuses slowly, the hanging drop concentrations surpasses saturation and becomes super saturated. Protein will begin to nucleate (clump up) and if correctly into a nice crystal lattice forming micro crystals. After nucleation and stabilization of microcrystal micro crystals accumulate more proteins to form larger crystals.

Slide 34: When hanging drops are clear, there is no precipitation. Slide 35: Precipitation has occured but possibly to fast as they are not in the form of crystals Slide 36: Slightly better, on the right track towards crystallization Slide 37: better structure by changing chemical nature of solution Slide 38 and 39 : Ideal crystallization. Depends on many factors. Protein purity and homogeneity -Purity refers contaminants interfere with the packing of the molecule in 3d space -Homogeneeity: SDS page for example shows the presence of one protein, however one may form monomers whereas others from multimers (aggregates). Precipitant Conditions -Molecules in the precipitant may help or prevent in the crystallization. Form bridges or interact at interface between proteins Salt concentration - Ions of salts can interact with charged amino acids and create bridges between them or in some cases prevent crystallization by competitively competing with binding of another protein pH - Affects the charges of proteins

Membrane proteins -More structures of cytosolic proteins have been studied -Less of membrane proteins because they are tougher to crystallize. Slide 49 (explanation of how membrane proteins are difficult to crystallize -membrane proteins only function when in the lipid layer. You must form micelles with detergent. You're crystallizing basically an entire micelle structure with protein embedded.

Lecture 5 X-ray II: Braggs law Collection of diffraction data - When X-rays bombard crystals, 95 % transmit through the protein (energy of photons is too great?) without interacting with electrons - 4-5 percent are scattered by the crystal (in all directions) - Some emerge out of phase ( interfere destructively ) no net detecting - Some emerge "in phase" leading to constructive or positive interference (detection of x-ray) Slide4Scattering of X-rays - X-rays hit proteins and interact with electrons of electron cloud of each amino acid in the protein - Explanation on slides is pretty good. Slide 5 and 6 - electrons emit x rays at same frequency of incident, but at angle (theta) - Question Important for midterm or exam: arrange the following atoms in terms of scattering ability ( ie which have the highest number of electrons ) Slide 7 - example of simple case where scattering pattern from two electron clouds interfere. Imagine instead scattering pattern from 500 amino acids. Slide 8 - Addition of waves - When in phase ( for example two sin curves) the resulting curve (sin(theta) + sin (theta) is 2sin(theta). Amplitude increases - the opposite is true fro out of phase (everything cancels out) Slide 11

- Mathematics that links position of molecule and point of constructive interference ( of brightness) - There are also region more brighter than others which give hints about structure - Diffraction pattern also depend on the packing of the crystal. Spots more separated, more packed crystal (larger theta angle) Braggs law As explain on slide 16. Important note must be made about "parallel planes" Form parallel planes going through the corners of unit cell like on slide 20 What dictates whether X-rays emerge in phase ( incident rays travel different distances depending on which plane they reflect off of, causing out of phase or in phase) Only when the extra distances traveled are integer multiples of wavelength

Summary on Slide 26 (exactly what is on the slide ): Need to know the equation considered as foundation of crystallography Distances between the same amino acid in protein is the same because of symmetry. Intensity and not position of spots tells us about the structure of the protein. This is due to interference from waves coming from different atoms that destructively and constructively intefere(Important) SLIDE 47 important excercise