lecture5_techniques2

7/29/2019 lecture5_techniques2

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Structural Analysis of ProteinStructureCircular Dicroism

Fluorescence

X-ray

NMR


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Methods for Secondary Structural Analysis

A number of experimental techniques can selectivelyexamine certain general aspects of macromolecularstructure with relatively little investment of time and

sample. Reasonable estimates of protein secondary structure

content can be determined empirically through the use of

Circular dichroism (CD) spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopyFT-infrared spectroscopy


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Circular Dichroism Circular dichroism (CD) spectroscopy is a form of light

absorption spectroscopy that measures the difference inabsorbance of right- and left-circularly polarized light (ratherthan the commonly used absorbance of isotropic light) by a

substance. It is measured with a CD spectropolarimeter. The instrument

needs to be able to measure accurately in the far UV atwavelengths down to 190 - 170 nm (170 - 260 nm).

The difference in left and right handed absorbance A(l)- A(r)is very small (usually in the range of 0.0001) corresponding toan ellipticity of a few 1/100th of a degree.


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Physics of

CD

Linear polarized light can be viewed as a superposition of

opposite circularly polarized light of equal amplitude and phase. A projection of the combined amplitudes perpendicular to the

propagation direction thus yields a line. When this light passes through an optically active sample with a

different absorbance A for the two components, the amplitudeof the stronger absorbed component will be smaller than that ofthe less absorbed component. The consequence is that aprojection of the resulting amplitude yields an ellipse instead ofthe usual line, while the polarization direction has not changed.

The occurrence of ellipticity is called Circular Dichroism.


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Rotation of Plane-polarized Light byan Optically Active Sample

Pockels cell produces a beam that is alternately switchedbetween L and R. The beam then passes through the

sample to a photomultiplier. The detected signal can thenbe processed as A vs .


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Physical Principles of CD

Inherently asymmetric chromophores (uncommon) orsymmetric chromophores in asymmetric environments willinteract differently with right- and left-circularly polarized

light resulting in circular dichroism. Right- and left-circularly polarized light will be absorbed

to different extents at some wavelengths due to differencesin extinction coefficients for the two polarized rays calledcircular dichroism (CD).

Circular dichroism can only occur within a normalabsorption band and thus requires either an inherentlyasymmetric chromophore (uncommon) or a symmetric onein an asymmetric environment.


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Instrumentation

The most common instruments around are thecurrently produced JASCO, JobinYvon, OLIS,

and AVIV models. We have the Jasco 710 and 810 models with

temperature controllers. The air cooled 150WXenon lamp does not necessitate water cooling.

You still need to purge with ample nitrogen to getto lower wavelengths (below 190 nm).


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Typical Initial Concentrations Protein Concentration: 0.5 mg/ml (The protein concentration

needs to be adjusted to produce the best data). Cell Path Length: 0.5-1.0 mm. If absorption poses a problem,cells with shorter path (0.1 mm) and a correspondingly increasedprotein concentration and longer scan time can be employed.

Stabilizers (Metal ions, etc.): minimum Buffer Concentration: 5 mM or as low as possible, while

maintaining protein stability. A typical buffer used in CDexperiments is 10 mM phosphate, although low concentrationsof Tris, perchlorate or borate is also acceptable.

As a general rule of thumb, one requires that the totalabsorbance of the cell, buffer, and protein be between 0.4 and1.0 (theoretically, 0.87 is optimal).

A spectra for secondary structure determination (260 - 178 nm)will require 30-60 minutes to record (plus an equivalent amountof time for a baseline as every CD spectrometer.


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Sample Preparation and Measurement Additives, buffers and stabilizing compounds: Any compound,

which absorbs in the region of interest, (250 - 190 nm) should beavoided. A buffer or detergent, imidazole or other chemical shouldnot be used unless it can be shown that the compound in questionwill not mask the protein signal.

Protein solution: The protein solution should contain only thosechemicals necessary to maintain protein stability/solubility, and at

the lowest concentrations possible. The protein itself should be aspure as possible, any additional protein will contribute to the CDsignal.

Contaminants: Particulate matter (scattering particles), anythingthat adds significant noise (or artificial signal contributions) to the

CD spectrum must be avoided. Filtering of the solutions (0.02 msyringe filters) may improve signal to noise ratio. Data collection: Initial experiments are useful to establish the best

conditions for the "real" experiment. Cells of 0.5 - 1.0 mm pathlength offer a good starting point.


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CD Data Analysis The difference in absorption to be measured is very small.

The differential absorption is usually a few 1/100ths to afew 1/10th of a percent, but it can be determined quiteaccurately. The raw data plotted on the chart recorder

represent the ellipticity of the sample in radians, which canbe easily converted into degrees


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CD Data Analysis To be able to compare these ellipticity values we need to

convert into a normalized value. The unit most commonlyused in protein and peptide work is the mean molarellipticity per residue. We need to consider path length l,

concentration c, molecular weight M and the number ofresidues.

in proper units (CD spectroscopists use decimol)

which finally reduces to

The values for mean molar ellipticityper residue are usually in the 10,000's


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CD Data Analysis

The molar ellipticity [] is related to the difference inextinction coefficients

[] = 3298 . Here [] has the standard units of degrees cm2 dmol -1

The molar ellipticity has the units degrees decilitersmol-1 decimeter-1.


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CD Signal of Proteins For proteins we will be mainly concerned with absorption

in the ultraviolet region of the spectrum from the peptidebonds (symmetric chromophores) and amino acidsidechains in proteins.

Protein chromophores can be divided into three classes: the

peptide bond, the amino acid sidechains, and anyprosthetic groups.

The lowest energy transition in the peptide chromophore isan n p* transition observed at 210 - 220 nm with very

weak intensity (emax~100).----p* p p* ~` 190 nm emax~7000----n n p 208-210, 191-193 nm emax~100----p


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Comparison of theUV absorbance

(left) and thecircular dichroism(right) of poly-L-lysine in differentsecondary structureconformations as afunction of pH.

The n p* transition appears in the a-helical form of thepolymer as a small shoulder near 220 nm on the tail of a muchstronger absorption band centered at 190 nm. This intense band,responsible for the majority of the peptide bond absorbance, is app* transition (emax ~ 7000).

Using CD, these different transitions are more clearly evident.Exciton splitting of the p p* transition results in the negative

band at 208 and positive band at 192 nm.


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CD Spectra of Proteins Different secondary structures of peptide bonds have

different relative intensity of n p* transitions, resultingin different CD spectra at far UV region (180 - 260 nm).

CD is very sensitive to the change in secondary structuresof proteins. CD is commonly used in monitoring the

conformational change of proteins. The CD spectrum is additive. The amplitude of CD curve

is a measure of the degree of asymmetry.

The helical content in peptides and proteins can be

estimated using CD signal at 222 nme222= 33,000 degrees cm2 dmol -1 res-1

Several curve fitting algorithms can be used to deconvoluterelative secondary structures of proteins using the CD

spectra of proteins with known structures.


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Protein CD Signal

The three aromatic side chains that occur in proteins (phenylgroup of Phe, phenolic group of Tyr, and indole group ofTrp) also have absorption bands in the ultraviolet spectrum.

However, in proteins, the contributions to the CD spectra inthe far UV (where secondary structural information islocated) is usually negligible. Aromatic residues, ifunusually abundant, can have significant effects on the CD

spectra in the region < 230 nm, complicating analysis. The disulfide group is an inherently asymmetric

chromophore as it prefers a gauche conformation with abroad CD absorption around 250 nm.


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Far UV CD Spectra of Proteins

[]x

10-3degreescm

2d

mol-1


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CD Spectra ofProtein Each of the threebasic secondary

structures of apolypeptide chain(helix, sheet, coil)show a characteristic

CD spectrum. Aprotein consisting ofthese elements shouldtherefore display a

spectrum that can bedeconvoluted into thethree individualcontributions.


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CD Spectra Fit

In a first approximation, a CD spectrum of a protein orpolypeptide can be treated as a sum of three components:a-helical, b-sheet, and random coil contributions to thespectrum.

At each wavelength, the ellipticity () of the spectrum will

contain a linear combination of these components:

(1)

T is the total measured susceptibility, h the contributionfrom helix, s for sheet, c for coil, and the correspondingthe fraction of this contribution.


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CD Spectra Fit As we have three unknowns in this equation, a

measurement at 3 points (different wavelengths) wouldsuffice to solve the problem for , the fraction of eachcontribution to the total measured signal.

We usually have many more data points available from ourmeasurement (e.g., a whole CD spectrum, sampled at 1 nmintervals from 190 to 250 nm). In this case, we can try tominimize the total deviation between all data points andcalculated model values. This is done by a minimization of

the sum of residuals squared (s.r.s.), which looks asfollows in our case :


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Using CD to Monitor 3 Structure of Proteins

CD bands in the near UV region (260350 nm) areobserved in a folded protein where aromatic sidechains areimmobilized in an asymmetric environment.

The CD of aromatic residues is very small in the absence of

ordered structure (e.g. short peptides). The signs, magnitudes, and wavelengths of aromatic CDbands cannot be calculated; they depend on the immediatestructural and electronic environment of the immobilizedchromophores.

The near-UV CD spectrum has very high sensitivity for thenative state of a protein. It can be used as a finger-print ofthe correctly folded conformation.


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Domain 1of CD2 CD2 is a cell adhesionmolecules.

Domain 1 of CD2 has a IgGfold. Nine b-strands form abeta-sandwich structure.

Two Trp residues, W-7 andW-32 (green) are located atthe exposed and buriedregion of the protein,respectively.

Our lab has used CD2 as amodel system to understandconformation flexibility of

proteins


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CD2 is Stable from pH 1 to 10

-3000

-2500

-2000

-1500

-1000

-500

0

500

1000

200 210 220 230 240 250 260

[]

(degcm

2d

mol

-1

res

-1

)

Wavelen th nm


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Conformational Change of CD2

-3000

-2000

-1000

0

200 210 220 230 240 250 260

[](degcm

2dm

ol-1)

Wavelength (nm)

c

85 C

25 C

6M GuHCl


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CD2 Becomes Significantly Helical in TFE

-2 104

-1.5 10

4

-1 104

-5000

0

5000

200 210 220 230 240 250 260

0% TFE10% TFE17% TFE19% TFE30% TFE80% TFE[

](degcm

2d

mol

-1r

es

-1)

Wav elength (nm)


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Near UV CD Spectra of CD2

CD2 lossesits nativewell packed

tertiarystructure athightemperature

and in 6MGuHCl

-400

-300

-200

-100

0

100

200

260 280 300 320 340 360

[](degcm

2d

mol-

1)

Wavelength (nm )

a

6 MGuHCl85 C

25 C


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CD2 losses its Tertiary Structure in TFE

-400

-300

-200

-100

0

100

200

260 270 280 290 300 310 320

0% TFE

10% TFE

17% TFE

30% TFE[](degcm

2d

m

ol-1

res

-1)

Wavelength (nm)


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Trp Fluorescence Emission Spectra of

CD2 under Different Conditions In a hydrophobic

environment (inside ofa folded protein), Trp

emission occurs atshorter wavelength.When it is exposed tosolvent, its emission is

very similar to that ofthe free Trp amino acid(red shift occurs).

0

1 104

2 104

3 104

4 104

300 320 340 360 380 400

Fluorescenc

ei

ntensity

Wavelength (nm)

c

Trp25C

6M GuHCl

85C


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Secondary Structure Prediction of CD2x-structure A B C C' C"

1 10 20 30 40 50Rat CD2 RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVAEFKRKMKPFLK PHD CCCCSSSSCCCCCSSSCCCCCCCCCCHHHHHHHHCCHHHHHHHHHCCCCSS GOR CCCCSSSSSSSCCCSCCCCCCCCCCCHCHSSHHHCCHHHHHHHHHHHHHHH SOPMA

CCCCSSHCCCCCCSSSCCCCCCCCCCCCHSSHHCCCSHHHHHHHHHHHHHC

x-structure D E F G

60 70 80 90Rat CD2 SGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILNKALDLRILE PHD CCCSSSSSCCCSSSCCCCCCCCCCSSSSSSCCCHHHHHHHHCCCCCCC GOR HHHHHHHHHHHHHHHSSSSCCCCSSSSSSSSCCCCSSHHHHHHHHHHH SOPMA CCCSSSSCCCCSSSSSSCCCCCCCSSSSSSSCCCCSSSSHHHHHSSHC

H = a-helix S = b-sheet C = coil

b-sheet 3 -helix


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S f CD


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Summary of CD Circular dichroism spectroscopy is used to gain information

about the secondary structure and folded state of proteins andpolypeptides in solution.

Benefits: Uses very little sample (200 ul of 0.5 mg/ml solutionin standard cells)Non-destructiveRelative changes due to influence of environment on

sample (pH, denaturants, temperature, etc.) can bemonitored accurately. Drawbacks: Interference with solvent absorption in the UV

region

Only very dilute, non-absorbing buffers allowmeasurements below 200 nmAbsolute measurements subject to a number ofexperimental errorsAverage accuracy of fits about +/- 10%

CD spectropolarimeter is relatively expensive


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X-ray Crystallography

X-rays are electromagnetic radiation atshort wavelengths, emitted when electrons

jump from a higher to a lower energy state.Growth of crystalsX-ray diffractionHeavy-metal complexBuild modelRefinement


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Drug design

information

http://www-structure.llnl.gov/xray/101index.html;http://www.aps.anl.gov/aps/frame_home.html

Crystallization

Data collection Data procession

Model refinement

Structure

analysisX-raycrystallography
http://www-structure.llnl.gov/xray/101index.htmlhttp://www-structure.llnl.gov/xray/101index.htmlhttp://www-structure.llnl.gov/xray/101index.htmlhttp://www-structure.llnl.gov/xray/101index.html


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Crystal

A crystal is built up from many billions of small identical units, or unitcells. These unit cells are packed against ach other in three dimensions,much as identical boxes are packed and stored in a warehouse. The unitcell may contain one or more than one molecule. Although the number ofmolecules per unit cell is always the same for all the unit cells of a singlecrystal, it may vary between different crystal forms of the same protein.

The diagram shows in two dimensions several identical unit cells, eachcontaining two objects packed against each other. The two objects withineach unit cell are related by twofold symmetry to illustrate that each unitcell in a protein crystal can contain several molecules that are related bysymmetry to each other.


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Each unit cell can containseveral molecules that arerelated by symmetry.

The diagram shows identicalblocks, each containing twoobjects packed against eachother.

Many small identical blocks or unit cellsare packed against other in 3D.

In order to obtain a crystal, moleculesmust assemble into a periodic lattice.

www.via.ecp.fr/~im/musee/escher.html
http://www.via.ecp.fr/~im/musee/escher.htmlhttp://www.via.ecp.fr/~im/musee/escher.html


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Crystals & X-ray Diffraction

Well-ordered protein crystals (a) diffract x-rays and producediffraction patterns that can be recorded on film (b) (Lauephotograph). The diffraction pattern was obtained usingpolychromatic radiation from a synchrotron source in thewavelength region 0.5 to 2.0 .

enzyme RuBisCo


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Protein Crystal Packing

Protein crystals contain largechannels and holes filled withsolvent molecules. Thesubunits (colored disks) form

octamers of molecular weightaround 300 kDa of glycolateoxidase, with a hole in themiddle of each of about 15 in diameter. Between the

molecules there are channels(white) ~ 70 in diameterthrough the crystal.


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The Hanging-drop Method of

ProteinCrystallization

About 10 ml of a 10 mg/ml protein solution in a buffer with addedprecipitant --- such as ammonium sulfate, at a concentration below that atwhich it causes the protein to precipitate --- is put on a thin glass plate thatis sealed upside down on the top of a small container. In the containerthere is about 1 ml of concentrated precipitant solution. Equilibriumbetween the drop and the container is slowly reached through vapordiffusion, the precipitant concentration in the drop is increased by loss ofwater to the reservoir, and once the saturation point is reached the proteinslowly comes out of solution. If other conditions such as pH and

temperature are conducive, protein crystals will form in the drop.


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A Diffraction Experiment

When the X-ray goes through the crystal, beams is diffracted and diffractionpattern is recorded on a detector. The crystal is rotated a certain degree whilethis pattern is recorded. A series of frames are collected.

Determine the size of the unit cell by Bragg's law:

2d sin = d= /(2* sin ).

http://www-structure.llnl.gov/Xray/101index.html


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iff i f


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Diffraction of X-rays by a Crystal

(a) When a beam of x-rays (red) shineson a crystal all atoms in the crystalscatter x-rays in all directions. Mostof these scattered x-rays cancel out,but in certain directions (blue arrow)they reinforce each other and add up toa diffracted beam. Different sets ofparallel planes (b) can be arrangedthrough the crystal so that each cornerof all unit cells is on one of the planesof the set. X-ray diffraction can be

regarded as reflection of the primarybeam from sets of parallel planes inthe crystal, separated by a distance d.The primary beam strikes the planes atan angle and the reflected beamleaves at the same angle, the reflection

angle.


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X-rays (red) that are reflected from the lower plane have traveledfarther than those from the upper plane by a distance BC + CD, whichis equal to 2dsin.

Reflection can only occur when this distance is equal to the wavelengthl of the x-ray beam and Bragg's law (2d sin = l). To determine thesize of the unit cell, the crystal is oriented in the beam so that reflectionis obtained from the specific set of planes in which any two adjacent

planes are separated by the length of one of the unit cell axes. Thisdistance, d, is then equal to l/(2sin). The wavelength, l, of the beamis known since we use monochromatic radiation. The reflection angle,, can be calculated from the position of the diffracted spot on the film,where the crystal to film distance can be easily measured. The crystal

is then reoriented, and the procedure is repeated for the other two axesof the unit cell.

Diffraction ofX-rays by a

Crystal

Th fl ti l f


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Diffraction of

X-ray Beams

The reflection angle, q, for adiffracted beam can be calculatedfrom the distance (r) between thediffracted spot on a film and the

position where the primary beamhits the film. From the geometryshown in the diagram, the tangentof the angle 2 = r/A. A is thedistance between crystal and filmthat can be measured on theexperimental equipment, while rcan be measured on the film.Hence, can be calculated. Theangle between the primary beamand the diffracted beam is 2, as

can be seen on the enlarged insertto the right. It shows that this angleis equal to the angle between theprimary beam and the reflectingplane plus the reflection angle, both

of which are equal to .

Properties of Diffracted Waves


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Properties of Diffracted Waves Two diffracted beams, each of which is defined by three properties:

amplitude, which is a measure of the strength of the beam andwhich is proportional to the intensity of the recorded spot,

phase, which is related to its interference, positive or negative,with other beams, and

wavelength, which is set by the x-ray source for monochromaticradiation.

We need to know all three properties to determine the position of theatoms giving rise to the diffracted beams.


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Multiple Isomorphous

Replacement (MIR)

Heavy atoms (strong diffraction) are introduced into theunit cell of the crystal to obtain phase information bysoaking crystals in the metal solution.

Intensity differences are used to deduce the positions of theheavy atoms in the crystal unit cell. Fourier summations ofthese intensity differences give Patterson maps of the

vectors between the heavy atoms. From the positions of the heavy atoms in the unit cell, we

can get amplitudes and phases. More than two different heavy-metal complexes are

needed to give a reasonably good phase determination for

all reflections.


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Building a Model The amplitude and phases of the diffraction data from the

protein crystals are used to calculate an electron-density-map of the repeating unit of the crystal.

This map is then interpreted as a polypeptide chain with a

particular amino acid sequence. The resolution (in ) is limited by the map error,resolution of the diffraction map.

At low resolution (5 or higher), the shape of themolecule can be obtained.

At medium resolution (~3 ), the trace of the polypeptidechain, i.e. active site, can be obtained

At high resolution ( 2 ), the a.a. sidechianscan beresolved.


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Electron-density maps at different resolutionshow more detail at higher resolution.

(d) 1.1


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Reducing Errors by Refinement

In the refinement process, the model is changed to minimizethe difference between the experimentally observeddiffraction amplitudes and those calculated for a

hypothetical crystal containing the model instead of the realmolecules.The difference is called the R factor, with 0.0 being exact

agreement and 0.59 total disagreement.0.15 < R < 0.20 = well determined structure

R ~ 0.30 = medium structureR > 0.30 = bad structure


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B-factorATOM 1 N PRO A 190 -0.567 24.363 16.753 49.28

ATOM 2 CA PRO A 190 -0.399 23.026 17.339 49.21

ATOM 3 C PRO A 190 -1.288 21.990 16.644 49.61

ATOM 4 O PRO A 190 -2.520 22.007 16.772 49.44

In the pdb file of x-ray structures, the atoms positions is givenby four numbers, three of them for coordinates and onequantity B, which is called the B-factor or temperature factor.

B < 20 = well defined regions

B > 40 = atoms have high flexibility


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NMR Spectroscopy

It is possible to determine the secondary structure of a proteinusing NMR techniques without determining the three-dimensional structure. NMR is potentially the most powerful ofall the methods available for prediction of secondary structure.Unlike secondary structure determinations by CD, which provideoverall secondary structure content (% helix, % sheet, etc.), usingNMR parameters, secondary structures are localized to specificsegments of the polypeptide chain.

However, obtaining secondary structure from NMR data requires

considerably more material (milligrams) and effort (requiressequence specific resonance assignments) than the otherspectroscopic techniques and is limited to proteins of molecularweight amenable to NMR investigation (< 35 - 40 kDa).


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NMR Spectroscopy

In the past 10 years, nuclear magnetic resonance (NMR)spectroscopy has proved itself as a potentially powerfulalternative to X-ray crystallography for the determination

of macromolecular three-dimensional structure. NMR hasthe advantage over crystallographic techniques in thatexperiments are performed in aqueous solution as opposedto a crystal lattice.

However, the physical principles that makes NMR

possible, limits the application of this technique tomacromolecules of less than 35 - 40 kDa. Fortunately, alarge number of globular proteins and most proteindomains fall into this molecular weight regime.


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Physical Principles of NMR Sub-atomic particles (e.g., proton, neutron, electron, etc.)

possess a characteristic called spin angular momentum. Fromquantum mechanics, each particle has a spin value of 1/2. Thecombination of multiple particles in the nucleus results in anoverall spin property for each atomic isotope. Those isotopeswith an even number of protons and neutrons will have zeromagnetic spin (e.g., He-4, C-12 and O-16). An odd number of

protons and an even number of neutrons (e.g., H-1, N-15, or F-19) or an odd number of neutrons and an even number of

protons (e.g., He-3, O-17 or Ca-41) result in an overall(multiple of 1/2) spin. Those isotopes with odd numbers of bothprotons and neutrons (e.g., H-2 or N-14) have more complexspin states and are less suitable for direct NMR observation inmacromolecules.

Ph i l P i i l f NMR


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Physical Principles of NMR Fortunately, each of the four most abundant elements in

biological material (H, C, N, and O) have at least onenaturally occurring isotope with non-zero nuclear spin, andin principle, can be observed using NMR.

The naturally occurring isotope of hydrogen, H-1, is presentat > 99 % abundance and forms the basis of the experimentsdescribed here. Other important NMR-active isotopesinclude C-13 and N-15 present at 1.1 and 0.4 % naturalabundance, respectively. The low natural abundance of thesetwo isotopes makes their observation difficult on commonly

isolated natural products. These two nuclei are however very extensively used for

larger (> 10 kDa) proteins, which can be isotopicallyenriched (to > 95 % if necessary) when cloned into systems

with high expression yields.


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Chemical Shifts In the presence of an external magnetic field, the spinangular momentum of nuclei with isotopes of overall non-

zero spin will undergo a cone-shaped rotation motion calledprecession. The rate (frequency) of precession for eachisotope is dependent on the strength of the external field and

is unique for each isotope. For example, in a magnetic field of a given strength (e.g.

14.1 Tesla) all protons in a molecule will have characteristicresonance frequencies (chemical shifts) within a dozen or soparts per million (ppm) of a constant value (e.g., 600.13MHz) characteristic of the particular nuclear type.

These slight differences are due to the type of atom theproton is bound to (e.g., C, N, O, or S) and the localchemical environment. Thus each proton should, in

principle, be characterized by a unique chemical shift.


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Chemical shifts Since the chemical shift of a nucleus is sensitive to the

environment, it should also contain structural information. Correlations between chemical shift tendencies and secondary

structures have been identified. The alpha proton of all 20naturally occurring amino acids has been shown to have a strongcorrelation with secondary structure. Wishart et al., (1992) have

produced a simple method for secondary structure determinationby analyzing the difference between the alpha proton chemicalshift for each residue and that reported for the same residue typein a "random coil" conformation. Helical segments havegroupings of alpha protons whose chemical shifts areconsistently less than the random coil values whereas betastrands had values consistently greater. In this way, thelocation of helix and strand segments are possible (and quitereliable) although the boundaries of the secondary structuralelements are not as well defined.


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Secondary Shifts Plot of the

differencesbetween theobserved alpha

proton chemicalshifts and thecorrespondingrandom coilvalues, d(Hanative)

- d(Harandom),versus the aminoacid sequence ofGlutaredoxin 3


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J coupling

Structural information from NMR experiments comeprimarily from through-bond (scalar or J coupling) or through

space (the nuclear Overhauser effect NOE) magnetizationtransfer between pairs of protons. J couplings between pairs of protons separated by three

covalent bonds can be measured. The value of a three-bond Jcoupling constant contains information about the interveningtorsion angle. This is called the Karplus relationship and hasthe form:

3J = A cos () +B cos2 () + Cwhere A, B, and C are empirically derived constants for each

type of coupling constant (e.g.,3

JHAHN or3

JHAHB).

J li


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J coupling

Shown above is the empirically-derived Karplus relationshipbetween the vicinal three-bond coupling constant 3JHNa and the

intervening torsion angle phi.


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Coupling Constants

The three-bond coupling constant between the intra-residualalpha and amide protons is the most useful for secondarystructure determinations as it can be directly related to the

backbone dihedral angle phi. right-handed alpha helix, phi = -57, 3JHAHN = 3.9 Hz

right handed 3.10 helix, phi = -60, 3JHAHN = 4.2 Hz

antiparallel beta sheet, phi = -139, 3JHAHN = 8.9 Hz

parallel beta sheet, phi = -119, 3JHAHN = 9.7 Hz left-handed alpha helix, phi = 57, 3JHAHN = 6.9 Hz

T di i l NMR S t


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Two-dimensional NMR Spectrum The peaks along the

diagonal correspond tothe 1D spectrum. Theoff-diagonal peaks inthis NOE spectrumrepresent interactionsbetween hydrogenatoms that are closerthan 5 to each otherin space. From such aspectrum, one can

obtain information onboth the secondaryand tertiary structuresof the protein.

COSY NMR Experiments


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COSY NMR Experiments

COSY NMR experimentsgive signals that correspondto hydrogen atoms that arecovalently connected throughone or two other atoms.

Since hydrogen atoms in two adjacent residues are covalently

connected through at least three other atoms (for instance,HCa-C'-NH), all COSY signals reveal interactions within the sameamino acid residue. These interactions are different for differenttypes of side chains. The NMR signals therefore give a "fingerprint"

of each amino acid. The diagram illustrates fingerprints (red) of

residues Ala and Ser.


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NOE


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NOE The other major source of structural information comes

from through space dipole-dipole coupling between twoprotons called the NOE. The intensity of a NOE isproportional to the inverse of the sixth power of thedistance separating the two protons and is usually observedif two protons are separated by < 5 . Thus, the NOE is a

sensitive probe of short intramolecular distances. NOEsare categorized according to the location of the two protonsinvolved in the interaction.

Intraresidual NOEs are between protons within the sameresidue, whereas sequential, medium, and long range

NOEs are between protons on residues sequentiallyadjacent, separated by 1, 2 or 3 residues, and separated byfour or more residues in the polypeptide sequence. Anetwork of these short inter-proton distances form thebackbone of three-dimensional structure determination by

NMR.


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Sequential

Assignment

Adjacent residues in the amino acid sequence of a proteincan be identified from NOE spectra. The H atom attached toresidue i + 1 (orange) is close to and interacts with (purple

arrows) the H atoms attached to N, Ca, and Cb of residue i(light green). These interactions give cross-peaks in theNOE spectrum that identify adjacent residues and are usedfor sequence-specific assignment of the amino acidfingerprints derived from a COSY spectrum.

NOE Regions of secondary structure in


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NOE a protein have specificinteractions between hydrogenatoms in sequentially nonadjacent

residues that give a characteristicpattern of cross-peaks in an NOEspectrum. In antiparallel b-sheetregions there are interactionsbetween Ca-H atoms of adjacent

strands (pink arrows), between N-H and Ca-H atoms (dark purplearrows), and between N-H atomsof adjacent strands (light purplearrows). The corresponding

pattern of cross-peaks in an NOEspectrum identifies the residuesthat form the antiparallel b sheet.Parallel b sheets and a helices areidentified in a similar way.

NOEs


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NOEs A number of short (< 5 ) distances are fairly unique to

secondary structural elements. alpha helices are characterized by short distances between

certain protons on sequentially neighboring residues (e.g.,between backbone amide protons, dNN, as well as between betaprotons of residue i and the amide protons of residue i+1, dbN.

Helical conformations result in short distances between thealpha proton of residue i and the amide proton of residues i+3and to a lesser extent i+4 and i+2. These i+2, i+3, and i+4NOEs are collectively referred to as medium range NOEs

NOEs connecting residues separated by more than 5 residues

are referred to as long range. Extended conformations (e.g.,beta strands) on the other hand, are characterized by shortsequential, daN, distances. The formation of sheets also resultin short distances between protons on adjacent strands (e.g.,daa and daN).

Amide Proton Exchange Rates


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Amide Proton Exchange Rates The regular hydrogen-bonded secondary structures "protect"

amide protons involved in them as evidenced by theirsignificantly reduced amide proton exchange rates with thesolvent (H2O). Although nearly all polypeptide amideprotons are involved in hydrogen bonds in a globular proteinthose in regular secondary structures appear to be longer-lived.

For example, after placing a lyophilized sample of BPTI into2H2O many amide protons are completely replaced withdeuterium within 1hr. Over the next several hours, the amide

protons in the N-terminal and then the C-terminal helix alsocompletely exchange. However, some amide protonsparticipating in the central antiparallel sheet are still presentafter some months.

Selection of Secondary Structural Segment


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y g

Sequential stretches of residues with consistent secondarystructure characteristics (NOEs, coupling constants, slowlyexchanging amide protons, and chemical shifts) provide a reliableindication of the location of these structural segments. However,

the boundaries of these segments are difficult to define precisely.

Survey of NMR-derived StructuralP t Ch t i i R d d G 3


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yParameters Characterizing Reduced Grx3

Shown above, amide proton exchange rates with solvent water (filleddiamonds) kNH < 0.02 min-1, coupling constants: 3JHNa (filledcircles) < 6.0 Hz and (open circles) > 7.0 Hz, and sequential backbone

dNN and daN NOE connectivities are classified as strong, weak, orabsent and are represented by the thickness (or absence) of a barconnecting the residues in question. Medium range NOEconnectivities daN (i, i+3) and (i, i+4) are drawn as line segmentsconnecting the residues contributing to the observed cross peak ifpresent.

NMR determined Protein Structures


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NMR-determined Protein Structures

The multiple-dimensionalNMR spectra used to derive anumber of distanceconstraints for differenthydrogen atoms along thepolypeptide chain of the C-terminal domain of acellulase. The diagramshows 10 superimposedstructures that all satisfy the

distance constraints equallywell. These structures are allquite similar since a largenumber of constraints wereexperimentally obtained.

lecture5_techniques2

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