Unformatted text preview: BCH210 Tutorial - Week 4 First midterm exam is Tuesday, Oct. 12th, sample exams are now online. Friday October 8th, 2010 X-ray crystallography Over 14 000 structures from a variety of organisms have been crystallized (but > 100 000 pr- in humans) Some are duplicates but with different ligands bound or from different species Less than 200 are membrane proteins Limiting step - obtaining good crystals Can give you the highest resolution of protein structure 2 X-ray Crystallography The crystal is a mosaic of millions of copies of the protein The image is average of space (many copies) and time (of expt). 3 Electron Density Maps Diffraction patterns give electron density maps Resolution is dependent on the quality/regularity of the crystal Can compare to known structures or by replacing some atoms as reference points to give (,) angles Need to refine the results to give a good `fit' 4 Different ways of representing a protein A - space filling model of complete molecule B - ball and stick model of backbone C - Ribbon diagram of secondary structure 5 Nuclear Magnetic Resonance Based on the magnetic property of nuclei Nuclei with odd numbers of protons or neutrons can be measured, most common-H-1, C-13, N-15 (more rare) Use a large, very strong magnet to align nuclei in a soluble protein and look at energy absorbed Absorbance is influenced by neighboring atoms See thousands of resonances, need to compare to standards and previous expts to give 2D structure - smaller proteins are better 6 Nuclear Magnetic Resonance H from C,, CH 3 1-3 0-1 H from C 4-5 NH 8 Aromatics 6-7.5 7 Nuclear Magnetic Resonance Determines the proximity of active atoms
8 Nuclear Magnetic resonance Larger proteins have very complicated spectra Easier to look at changes in spectra under various circumstances, ie. which atoms are changing/moving 9 Infrared Spectroscopy (1500-1800 cm-1) Molecules rotate or vibrate at a specific frequencies Hit sample with infrared light and see which wavelengths are absorbed 10 Infra-red active covalent bonds Stretching tends to give stronger peaks 11 Infrared Absorption of Proteins Amide 1: 1640 - 1680 cm-1 C=O stretch (80%) Minor N-H bend (20%) Band position gives / secondary structure H bonds affect this band Amide 2: 1530 - 1550 cm-1 12 C-N stretch (60%) C-N-H bend (40%) Secondary structure using IR Can estimate secondary structure due to H bonds by inspecting the frequencies at which the amide I bonds absorb infrared radiation. The area under the peak provides info about structure abundance. Stronger H bonds weaken C=O resulting in lower energy needed to stretch. 13 Secondary structure using IR turn >1670 cm-1 random a helix 1650-1660 cm-1 random 1640-1650 cm-1 b sheet 1620-1640 cm-1 aggregated/denatured <1620 cm-1 14 Infrared spectroscopy chromophores X-H C-H Alcohol, amine, amide COOH, saturated/unsaturated carbons Amide I, II C=O 15 IR question 16 Circular Dichroism (190-250 nm) Based on the absorption of polarized light Measured in the ultraviolet range Gives helix, sheet or random structure info Best for looking at if a protein is folded, or changes due to mutation or ligand binding 17 Circular Dichroism Chiral molecules absorb left and right circularly, polarized UV light 18 Circular Dichroism Melting changes it's secondary structure 19 Protein structure question 20 Protein Fluorescence The ability to absorb light and emit it at a longer wavelength Protein fluorescence is a sum of all the fluorescent AAs in a protein 3 aromatic amino acids contribute to fluorescence Tryptophan - strongest (indole ring) Tyrosine - weaker emitter (25% of Trp) Phenylalanine - only weakly fluorescent 21 Tryptophan fluorescence 22 It's midterm time! 25 M/C, 4 options, 25% Student card Pencil Eraser Calculator Make sure you go to the correct room! 23 Retention Average Retention Rate After 24 Hours 5% 10% 20% 30% 50% 75% 90% The diagram shows the average percentage of retention of material after 24 hours for each of the instruction methods. Note that the percentages are not additive. Source: Adapted from National Training Laboratories of Bethel and NTL Institute of Alexandria, VA, cited in David Sous (2006) , How the Brain Learns, 3rd ed. California: Corwin Press. Reithmeier section BCH210 is picking up in pace 6 lectures packed into 5 Lots of details, but not a lot of words on his slides! Importance of being in class... Make connections between the material Check your notes with a friend 25 Phospholipids Contain either a glycerol or sphingosine backbone 26 Lipid Head Groups Head groups are attached to C3 via a phosphodiester bond Phosphate is -ve, lipids can be zwitterionic or charged depending on functional group 27 Acyl Chains Acyl chains can vary in length, mostly even # of carbons Acyl chains can be saturated (usually C1) or unsaturated (C2) (double bonds) Abbreviated as: C18:1, #carbons:# C=C 28 Steroid lipids Cholesterol is an essential component of membranes Also has a polar OH and hydrophobic rings and acyl chain Neutral lipid 29 Fatty Acids - Eicosanoids Consist of an acyl chain and a carboxylic acid at one end Also known as omega 6 fatty acids, 1st C=C 6 carbons from the end Most common is arachidonic acid (like a peanut!) Other hormones are derived from these, this makes them essential fatty acids 30 Lipids in solution Hydrophobic tails tend to associate away from water Can either form micelles, a bilayer or liposome Molecules can be trapped inside a liposome 31 Membrane Bilayer Lipids may move laterally in the plane of a liquid crystalline bilayer Flip flop is extremely rare, polar head groups need to cross the hydrophobic core Flippase are enzymes that can flip lipids across PC&SM - outer leaflet PS&PE - inner leaflet 32 Melting Temperature The transition from a gel to a liquid crystalline state Longer chains and saturated acyl chains melt at higher temperatures Cholesterol also helps maintain fluidity (less packed) 33 Melting Temperature Question How would you expect the type of lipids from different parts of the leg of a reindeer to vary in fatty acid composition? 34 Membrane Protein & Lipid Diffusion Can be demonstrated using mixing experiments or by photobleaching file:///Volumes/BI3_1/Lessons/12-3c_FluidMosaic-b3/ FluidMosaic.htm (voet&voet cd) Lipids may also redistribute (PS with Ca2+) http:// web.virginia.edu/Heidi/chapter9/Flash/figure9_8.html 35 Red Blood Cells RBC shape is determined by cytoskeleton, membrane protein movement is restricted by this 36 Lipids and membrane surprise! More fun stuff with the appearance of a biochemistry super star! 37 ...
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This note was uploaded on 11/23/2010 for the course BCH BCH210 taught by Professor Deber during the Fall '08 term at University of Toronto.
- Fall '08