bibc 100 handout 10

BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm

TA: Lydiesther Martinez [email protected] Section: Mon

WEEK 10 FINAL COMMENTS Every chapter has conclusion, make sure you know all the details Go back through the chapter and look at the figures (refer to slides), be able to interpret them Look at practice exams Ask yourself, what did I learn?

Determining Protein Structure X-Ray Diffraction Interactions of x-rays with electrons in molecules in a crystal NMR- Nuclear Magnetic Resonance Interactions of magnetic field (external) with the intrinsic magnetic properties of atomic nuclei which possess a spin angular momentum Both have things in common and discrepancies that tell you you need both Both req interaction of energy with material X-RAY Protein Crystal Virtually any size, small molecule or entire virus Few, high quality crystals Result: Electron-density map atomic model [[e- density map used to build atomic model ]] Requires: Phase determination of the diffracted beams for which heavy metals are used (needs infusion into crystals (heavy metals) NMR Spectroscopy Limited (2009) ~ 30,000 Da. (Limitation) Protein in High Conc. Result: Distance constraints between 1H atoms 3-D molecular model Requires: Isotopes (1H, 13C, 15N) Both req in protein in high concentration. Both are model dependent X-RAY Frozen Structure, trapped in that conformation in that crystal, so when you have dynamic aspects you need to obtain different crystals in different conformations NMR Time Resolved Dynamic Folding Studying in solution so you can see dynamics (folding problem, intermediates)



BOTH OF THEM NEED: Purified Protein Amino Acid Sequence Computers: Molecular Mechanics Molecular Dynamics Molecular Graphics Recombinant DNA Technology, allows you to purify prot in high quant, can introduce mutations (barnase t4lysozyme, diff things mutated to asses stability)

CRYSTALS (crystallography) Built up of billions of identical units the unti cell Figure from book (orange/yellow cell looking things ) No order no crystal Disorder low quality image

1. Well-Ordered 2. Large Size (~ 0.5 mm), size coned good 3. Pure protein at high concentration 1. in effort to crystallize, taking it out of solution, you want it to be around many prot neighbors 2. one path way is to take prot and concentrate it resulting in amorphous or get another crystal , most of the time results in aggregate. This is a drain of energy time and material because result in non prod crystal 3. whole point is to reduce protein solubility 4. change pH, wtc, prot [] in key HOW: Solution Aggregate Amorphous OR Ordered crystals Reducing Protein Solubility CRYSTALS Methods two techniques giving you crystals (discourage getting aggregates) (today we do it by robotics, if we want to change pH you try infinitesimal changes of pH so in screening condition can try thousands at a time. Same for temperature Hanging-Drop: Increase Conc. By vapor diffusion Microdialysis: Increase aggregation by loss of solvent Variables pH Temperature Gravity [protein] Solvent REQUIREMENTS



Crystals The repeating unit of a crystal, corresponding ~ to the volume occupied by a single molecule is called a unit cell A crystal is built by billions of identical unit cell X-Rays Electromagnetic radiation of wavelength 1.54 They are produced by a beam of accelerating e- on a copper anode target High E to low E diagram Accel e- heat at anode and result in X-ray FIGURE Vapor Diffusion

Ex: use hemoglobin, get droplet of blood on glass plate. Cover a flask, apply sealant so you have tight seal between prot sol of glass plate and material inside beaker. Causes precipitant, high [] of salt. If you take ammoni salt in high concentration precip, but low concentration this highly concentrated salt will try to extract water from prot solution. Will generate equil condition. Water will go from abundant source to less abundant source, sol of precip. This will allow slow water diffusion. VAPOR DIFFUSION after a few hours and days on surface you will see prot crystallized Protein crystals contain a lot of water because crystallized from water solution so tend to be soft. Way to know if you have crystal is to probe with glass capillary. Touch protein crystal and it will bend and not break with prot crystal soft. If same with sod cl crystal, capillary will break. test to orient you to the fact that crystals cont lots of water. FIGURE Xray diffraction X-rays used because the are electromagnetic radiation. Dist interested in is 1.54A. ( close to H bonds) But when Bombard crystal with beam Radiation destroys crystal

DETECTORS



a. b.

c.

Radiation destroys crystals i.e. Protection cooling, rotation (only surface layer destroyed) , brief exposure (req high intensity beam), large area detectors ( ucsd) Records of diffracted (scattered) beams are obtained on - film (x-ray): blackening of emulsion - electronic, solid state, detectors - large area detectors (electronic counters) UCSD Comparison of diffraction pattern of native protein crystal with complex of protein and a heavy metal (relying on the hevy metal not alterin conformation of protein)

FIGURE Diffraction picture In center you have burn region from xray source. Diffracted beams on side. Intensity of beam fades out from distance from center diffraction picture. How do you get from spots to prot structure, dep on following Law:

Braggs Law: (he was director in laboratory (Hb structure found) principles of diffraction could be extrapolated for larger prot. 2dsin = d = distance = reflection angle = wavelength, Dictates the conditions for diffraction, all spots related to law.

FIGURE Collection of unit cells. In pink you have xray beam in blue diffracted. Two molecules in grid. Beam enters and some diffracted. Beam hits top layer and diffracted accordingly. The other will enter a different layer. Both are diffracted beams. Trig tell you dist beams same size with addition of BC. Distance of unit cell, d, related to both distances. Q is how do you determine theta.



FIGURE Burning in center of fild creates point. Can measure dist R between spot and center. Known distance of where everything was placed. (more math ) x_x Now you have all the numbers that you need.

FIGURE

Waves have properties, amplitude and wavelength. They are in two diff phases. Crest of both at diff positions. Phase and amplitude are characteristics of waves tat you need to determine

Phase determination problem Each diffracted beam is defined by: Amplitude = intensity of spot-measure ( in dot diagram) Wavelength = you know Phase = lost in experiment Microscope out of focus/no eyepiece If take microscope and put ant and see perfectly well eyes, legs, etc. but then you turn around and take out oculars, you know ant is still there but everything is blurred. We are out of focus without the oculars KINEMAGE: red spots are water molecules. Prot shows is scorpion toxin at high res Prot crystals aka water prot crystals. You can diffuse heavy metals into them Side chains can be determined because of high resolution Solution: Multiple isomorphous replacement Diffusion of heavy metals into channels present in proteins (not K channel, these are cavities in prot crystals (soft related to presence of water) SH groups reactivity (heavy metals react with them, create lamp posts (mercury and Pt+) identifies where you are in the sequence



FIGURE

Replacement of light metals

Fourier summations of the intensity difference between diffracted spots from crystals of protein alone and 9prot + heavy metals] give vector maps between the heavy atoms. Here are THREE heavy atom. If take prot with thousands itll be much more difficult

FIGURE

Why heavy metals? If look at diagram both have same color code. Top: prot and heavy metal prod constructive BOTTOM: destructive interference, . Both condition show adv of heavy metal. At spec positions they interact.

Image is formed by applying a mathematical relation Fourier Transform Spot Wave of e- density Ampli comes from intensiryt. phase from heavy atom \ Purpose to use two different heavy metals to decrease ambiguity Difference Patterson Map To determine position of heavy atom in crystal To determine phase of heavy atom in crystal Use 2 different heavy atoms to decrease ambiguity

Calculation of the electron density map Interpretation of the electron density map Resolution: Quality of crystal (~2 ) 5-6 : Course of polypeptide chain, poor resolution, will only see backbone at best



FIGURE

~3 : side chains, e.g. ~ 2 : side chains 1-1.5 : atoms, aim

If take pic of building at high resolution you will see details of building

FIGURE

H to

ard to fit structure into density. Better resolution better idea of protein atoms/sidechains. Improving reslolution. If read any paper on crystallography, imp say at what resolution it was viewed. 5A is poor. 1A is good

Refinement model building R factor: residual disagreement, between a hypothetical crystal containing the model and the experimentally determined crystal. R = 0 perfect agreement R = 0.59 total disagreement For a resolution of 3 and R 0.3 not so good For 2, R=0.2 OK

NMR Atomic Nuclei (1H or 15N) possess an intrinsic spin angular momentum, resulting in a magnetic moment m that can interact with an externally applied magnetic field B Reason he told you about the buble place by parking lot : there are big magnets, isolated and shielded because have to be at exact precision. Used to spin angular momentum. The bigger the magnet the higher the resolution . In a B field the spins of H align Equilibrium alignment can be perturbed by pulses of radiofrequency (RF)



Can look at nuclei recover. Relaxation to equilibrium emits RF that can be measured

R B E N CLow [ High F n u h w v c e i i l m t r e i h o u c n s a r m l e e s n h p t i e f c t s t o a S p r e c f i e f r i e c n c e ]



Go from low to hight with high mag field, when coming down it will emit radio frequency causing a chemical shift. Which are specificit. Tells you two things, what nucleus and what environment (remember mportance of environment, NMR is a reporter of that) For Unique Assignments: Multi-Dimensional NMR: 2-D, 3-D, 4-D FIGURE

Concept: E increasing vs magnetic field strength. The higher the B the higher the E spin.

NMR 2-D NMR Diagonal: ~ 1-D spectrum Peaks off-diagonal : (cross-peaks) Interactions of H atoms that are close to each other in space COSY: (Correlation) Fingerprint of a.a. Distance between BONDED H atoms ( three chemical bonds, I.e. within the same a.a.) NOESY (NOE, Nuclear Overhausser Effect) Distance between H atoms close together in space ( 5 ) FIGURE -NMR 1D This one dimensional spectruem refers to ethanol, simple. Each peak rep diff H environments.

FIGURE Complex spectrum, simple peptide

This is not a protein, a small peptide. 22 or 23 AA. Complex diagram Who knows what this is so you need assistance, how to decode this messy spectrum into



constituent parts, or Cterminunus N terminus, AA how do you decoded AA composition 2. sequence, 3. 3d structure

INTERPRETATION Sequence Specific Assignment or Sequential assignment 1. COSY Cross peak unique for each amino acid, fingerprint a. Which a.a. in sequence? 2. NOESY a. interactions in space b. interactions of residues that are sequentially adjacent (i (AA) and i(AA)+1) i. ex. Alanine in 22, what AA is in 21 and 23? Will know if you know sequence 3. Amino Acid Sequence 4. Distance Constraints: for H atoms in i to H atoms in j 5. Structure Refinements a. computer modeling b. No unique structure but different structures that are compatible with data (ambiguity) though still use ambiguity to study prot prot interactions FIGURE Bottom part of alanine. All hydrogen define specific distances. Bottom part of ser and ala look to have similar hydrogen distances. However the H on top of the O in ser not the same distance so use COSY to differentiate them. Because theyre less than 3 chemical bonds. The top H of Ser from the bottom too far. Alpha helices loop: two helices are antiparralel. In linear seq first and ast H sep by huge sequence but in 3d its 5A apart. Space less than 5A allows you to see interactions SEE why importance NMR in folding. When prot unfold more than 5A apart so will not interact and will not have signal

FIGURE

B-sheet. Look at I and i+1 (I refers to AA) you see distance between their hydrogens are less than 5A and they are different. In 3D beta sheet, interactionsbetween intermolecular strands and far more apart in 1D. you can ident sequential residue if have sequence.

FIGURE



Proton chemical shift and see helical example. 2 and 5 close in 3D. in 3d graph you will see them in pink

FIGURE

2D NMR, diagonal equal to one dimensional spectrum. crox pic??? these spots are all unique, is it an alanine, what position is it in. This is whats called COSY

FIGURE NMR average of structures obtained

In NMR you dont get one structure, you get an average. What you see here is overlay. 10 or 12 structure. You know that there are many because you can count them. In overlay it looks very tight: telling you that because you are in solution you are not frozen so you get more possible conformation. Then get the average. Where do the structure differ?? Dont differ in the middle. Meaning that fram of structure are prob loops because tend to be mobile. They are in solutioin so readily if you have assay interacting with those regions, frames will be reduced because intereaction with another protein. KINEMAGE : zinc finger



8 structures total. You dont get a singular structure you get an average.

Combination of x-ray and NMR In general agreement is excellent with minor discrepancies. Structure Determination Complementary In general agreement (except minor discrepancies especially loops) Both require: Biochemical Information Supercomputer Processing Both differ and are complimentary, both dep on AA se mol modeling to see 3D structure. COMPLEMENTARY

Things to think about and should know, though you arent experts: When you hear a 1.5A crystallography, what does it mean? What does COSY NOSEY mean? What information can you obtain with this?

bibc 100 handout 10

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