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)
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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.
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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)
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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 ]
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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
BIBC 100 Structural Biochemistry Dr. Montal Spring 11 7pm
TA: Lydiesther Martinez [email protected] Section: Mon
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?