Identification of Secondary Structure：二级结构的识别

1、Structural Analysis of Protein StructureCircular DicroismFluorescenceX-rayNMRMethods for Secondary Structural Analysis A number of experimental techniques can selectively examine certain general aspects of macromolecular structure with relatively little investment of time and sample. Reasonable esti

2、mates of protein secondary structure content and structure change can be determined empirically through the use of Circular dichroism (CD) spectroscopy Fluorescence spectroscopyNuclear Magnetic Resonance (NMR) spectroscopyFT-infrared spectroscopyCircular Dichroism Circular dichroism (CD) spectroscop

3、y is a form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than 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

4、accurately in the far UV at wavelengths 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 to an ellipticity of a few 1/100th of a degree. Rotation of Plane-polarized Light by an Optically Ac

5、tive Sample Pockels cell produces a beam that is alternately switched between L and R. The beam then passes through the sample to a photomultiplier. The detected signal can then be processed as A vs .Instrumentation The most common instruments around are the currently produced JASCO, JobinYvon, OLIS

6、, and AVIV models. We have the Jasco 710 and 810 models with temperature controllers. The air cooled 150W Xenon lamp does not necessitate water cooling. You still need to purge with ample nitrogen to get to lower wavelengths (below 190 nm). Typical Initial Concentrations Protein Concentration: 0.5 m

7、g/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 increased protein concentration and longer scan time can be employed. Stabilizers (Metal ions, etc.): mi

8、nimum Buffer Concentration: 5 mM or as low as possible, while maintaining protein stability. A typical buffer used in CD experiments is 10 mM phosphate, although low concentrations of Tris, perchlorate or borate is also acceptable. As a general rule of thumb, one requires that the total absorbance o

9、f the cell, buffer, and protein be between 0.4 and 1.0 (theoretically, 0.87 is optimal). A spectra for secondary structure determination (260 - 178 nm) will require 30-60 minutes to record (plus an equivalent amount of time for a baseline as every CD spectrometer. Sample Preparation and Measurement

10、Additives, buffers and stabilizing compounds: Any compound, which absorbs in the region of interest, (250 - 190 nm) should be avoided. A buffer or detergent, imidazole or other chemical should not be used unless it can be shown that the compound in question will not mask the protein signal. Protein

11、solution: The protein solution should contain only those chemicals necessary to maintain protein stability/solubility, and at the lowest concentrations possible. The protein itself should be as pure as possible, any additional protein will contribute to the CD signal. Contaminants: Particulate matte

12、r (scattering particles), anything that adds significant noise (or artificial signal contributions) to the CD spectrum must be avoided. Filtering of the solutions (0.02 m syringe filters) may improve signal to noise ratio. Data collection: Initial experiments are useful to establish the best conditi

13、ons for the real experiment. Cells of 0.5 - 1.0 mm path length offer a good starting point.CD Data Analysis The difference in absorption to be measured is very small. The differential absorption is usually a few 1/100ths to a few 1/10th of a percent, but it can be determined quite accurately. The ra

14、w data plotted on the chart recorder represent the ellipticity of the sample in radians, which can be easily converted into degrees (radians)(degrees)CD Data Analysis To be able to compare these ellipticity values we need to convert into a normalized value. The unit most commonly used in protein and

15、 peptide work is the mean molar ellipticity per residue. We need to consider path length l, concentration c, molecular weight M and the number of residues.in proper units (CD spectroscopists use decimol)which finally reduces toThe values for mean molar ellipticity per residue are usually in the 10,0

16、00s CD Data Analysis The molar ellipticity is related to the difference in extinction coefficients = 3298 . Here has the standard units of degrees cm2 dmol -1 The molar ellipticity has the units degrees deciliters mol-1 decimeter-1.Circular Dichroism of Proteins It has been shown that CD spectra bet

17、ween 260 and approximately 180 nm can be analyzed for the different secondary structural types: alpha helix, parallel and anti-parallel beta sheets, turns, and other. A number of excellent review articles are available describing the technique and its application (Woody, 1985 and Johnson, 1990). Mod

18、ern secondary structure determination by CD are reported to achieve accuracies of 0.97 for helices, 0.75 for beta sheet, 0.50 for turns, and 0.89 for other structure types (Manavalan & Johnson, 1987).CD Signal of Proteins For proteins we will be mainly concerned with absorption in the ultraviole

19、t region of the spectrum from the peptide bonds (symmetric chromophores) and amino acid sidechains in proteins. Protein chromophores can be divided into three classes: the peptide bond, the amino acid sidechains, and any prosthetic groups. The lowest energy transition in the peptide chromophore is a

20、n n p* transition observed at 210 - 220 nm with very weak intensity (emax100).-p* p p* 190 nm emax7000-nn p* 208-210, 191-193 nm emax100-pComparison of the UV absorbance (left) and the circular dichroism (right) of poly-L-lysine in different secondary structure conformations as a function of pH. The

21、 n p* transition appears in the a-helical form of the polymer as a small shoulder near 220 nm on the tail of a much stronger absorption band centered at 190 nm. This intense band, responsible for the majority of the peptide bond absorbance, is a p p* transition (emax 7000). Using CD, these different

22、 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.CD Spectra of Proteins Different secondary structures of peptide bonds have different relative intensity of n p* transitions, resulting in different CD spec

23、tra at far UV region (180 - 260 nm). CD is very sensitive to the change in secondary structures of 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

24、 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 deconvolute relative secondary structures of proteins using the CD spectra of proteins with known structures. Protein CD Signal The three aromatic s

25、ide chains that occur in proteins (phenyl group of Phe, phenolic group of Tyr, and indole group of Trp) also have absorption bands in the ultraviolet spectrum. However, in proteins, the contributions to the CD spectra in the far UV (where secondary structural information is located) is usually negli

26、gible. Aromatic residues, if unusually abundant, can have significant effects on the CD spectra in the region vemor lex lemVibration levelsXe lampFluorescence measurementlex (nm)lem (nm)e (M-1 cm-1)YTrp (W)28034856000.2Tyr (Y)27430314900.1Phe (F)2572822400.04Trp Fluorescence Emission Spectra of CD2

27、under Different Conditions In a hydrophobic environment (inside of a folded protein), Trp emission occurs at shorter wavelength. When it is exposed to solvent, its emission is very similar to that of the free Trp amino acid (red shift occurs).01 1042 1043 1044 104300320340360380400Fluorescence inten

28、sityWavelength (nm)cTrp25C6M GuHCl85C5 1051 1061.5 1062 106270275280285290Fluorescence IntensityWavelength (nm)0.000.200.400.600.801.0010-610-5T26D-CaMwt-CaMN60D-CaMNormalized fluorescence intenistyCa2+free,MPheex = 254 nm8 1041.6 1052.4 1053.2 1054 1054.8 105300312.5325337.5Fluorescence intensityWa

29、velength (nm)0.000.200.400.600.801.0010-710-610-5N97D-CaMwt-CaMQ135D-CaMNormalized fluorescence intensityCa2+free,MTyrex = 277 nmInteraction between calmodulin and Ca2+By JasmineFluorescence resonance energy transfer (FRET) between donor and acceptor25 mM PIPES100 mM KClpH 6.8lex = 278 nmP = 1 M05 1

30、041 1051.5 1052 105500520540560580600EMOC-N85, 07-30-20091 uM EMOC-N852 uM Tb6 uM10 uM20 uM40 uM100 uM200 uM300 uMIntensityWavelength, nmA00.81050100150200Tb-binding comparison of EMOC-N85 and its variantsEMOC-N85EMOC-N85m1EMOC-N85m2Normalized IntensityTb, uMy = (1.0+M0+M1)-sqrt(1.0+M.Error

31、Value2.570412.149m1 NA0.048428ChisqNA0.96449Ry = (1.0+M0+M1)-sqrt(1.0+M.ErrorValue0.921048.2181m1 NA0.013584ChisqNA0.991Ry = (1.0+M0+M1)-sqrt(1.0+M.ErrorValue5.758329.155m1 NA0.036276ChisqNA0.98039RBFluorescence resonance energy transfer (FRET) between EMOC-N85 and Tb3+FRET fluorescence spectra betw

32、een EMOC-N85 and Tb3+ (A) and its curve fitting (B) of a Tb3+ titration. The interaction between Tb3+ and EMOC-N85 variants was indicated with different Tb3+-binding affinities (Kd) 12.1 M (EMOC-N85): 8.2 M (EMOC-N85m1); 29.2 M (EMOC-N85m2), respectively.11 stranded antiparallel b-barrel, a single c

33、entral a-helixSeveral loops and short helices capping the barrel on each endA chromophore formed by the residues 65-67 buried in the center of the barrelSingle-chain, 238 residues, 27 kDa, a soluble globular proteinResidues of 7-229 are essential for the fluorescent property of GFPA. Ribbon diagram

34、of the WT GFP structure taken from Proc. Natl. Acad. Sci. USA, (1997), 94, 2306-2311. The a-helices are shown in red, the b-strands are shown in green, and the chromophore is shown as a ball-and-stick model.B. An example of GFP in the natural environment of jellyfish in the ocean, taken from http:/w

35、ww.plantscicam.ac.uk/haseloff /GFP/GFPbackgrnd.html/GFP becomes one of the most popular and exciting new technologies in biochemistry and cell biologyABEGFP-based Ca2+ sensors with different Ca2+-binding affinities are developed by two different approachesHypothesisEGFP-based Ca2+ sensors with diffe

36、rent Ca2+ affinities in the range of 0.1 mM - 5 mM can be developed by creating a Ca2+ binding site into the chromophore sensitive locations of fluorescent proteins.7891011123456N-terminalC-terminalEGFPGraftingDesignCa2+ binding responseSpectroscopic characterization of Ca2+ sensor Ca-G1-37. (A) Vis

37、ible absorption spectrum for sensor Ca-G1-37 with increasing Ca2+ concentrations. Ca2+ dependence of fluorescence emission spectra with excitation of lex = 398 nm (B) and lex = 490 nm (C). Symbols of different Ca2+ concentrations in (B) and (C) are same as that in (A). The measurements were performe

38、d at 17 M Ca-G1-37 for visible absorption and 1.7 M Ca-G1-37 for fluorescence experiments with 10 mM Tris and 1 mM DTT (pH 7.4), respectively. The arrows indicate the direction of signal change resulting from an increase in the Ca2+ concentration. (D) Normalized F(398nm)/F(490nm) ratio curve-fitting

39、 of the Ca2+ titration data.Ex 398 nmEx 490 nmSummary of fluorescence Fluorescence is the emission of radiation that occurs when a molecule in an excited electronic state returns to the ground state. Application: Fluorescence has an important role in the structural determinants of proteins, DNA or R

40、NA, etc. Advantages: Small sample volumes (800L 3mL) Low concentration (0.1 5 M) Short experiment time (10-60 minute) Short data analysis time (5-30 minute) Recovery of sample Disadvantages: Large Stokes Shift Background fluorescence (Impurities in buffers and autofluorescence in cells) Scattered li

41、ght (problem with cloudy samples)X-ray Crystallography X-rays are electromagnetic radiation at short wavelengths, emitted when electrons jump from a higher to a lower energy state. Growth of crystals X-ray diffraction Heavy-metal complex Build model Refinement Drug design information http:/www-struc

42、/xray/101index.html; /aps/frame_home.htmlCrystallizationData collectionData processionModel refinementStructure analysis X-ray crystallographyCrystalA crystal is built up from many billions of small identical units, or unit cells. These unit cells are packed against

43、 ach other in three dimensions, much as identical boxes are packed and stored in a warehouse. The unit cell may contain one or more than one molecule. Although the number of molecules per unit cell is always the same for all the unit cells of a single crystal, it may vary between different crystal f

44、orms of the same protein. The diagram shows in two dimensions several identical unit cells, each containing two objects packed against each other. The two objects within each unit cell are related by twofold symmetry to illustrate that each unit cell in a protein crystal can contain several molecule

45、s that are related by symmetry to each other. Each unit cell can contain several molecules that are related by symmetry.The diagram shows identical blocks, each containing two objects packed against each other. Many small identical blocks or unit cells are packed against other in 3D.In order to obta

46、in a crystal, molecules must assemble into a periodic lattice. www.via.ecp.fr/im/musee/escher.html Crystals & X-ray Diffraction Well-ordered protein crystals (a) diffract x-rays and produce diffraction patterns that can be recorded on film (b) (Laue photograph). The diffraction pattern was obtai

47、ned using polychromatic radiation from a synchrotron source in the wavelength region 0.5 to 2.0 .enzyme RuBisCoProtein Crystal PackingProtein crystals contain large channels and holes filled with solvent molecules. The subunits (colored disks) form octamers of molecular weight around 300 kDa of glyc

48、olate oxidase, with a hole in the middle of each of about 15 in diameter. Between the molecules there are channels (white) 70 in diameter through the crystal.The Hanging-drop Method of Protein CrystallizationAbout 10 ml of a 10 mg/ml protein solution in a buffer with added precipitant - such as ammo

49、nium sulfate, at a concentration below that at which it causes the protein to precipitate - is put on a thin glass plate that is sealed upside down on the top of a small container. In the container there is about 1 ml of concentrated precipitant solution. Equilibrium between the drop and the contain

50、er is slowly reached through vapor diffusion, the precipitant concentration in the drop is increased by loss of water to the reservoir, and once the saturation point is reached the protein slowly comes out of solution. If other conditions such as pH and temperature are conducive, protein crystals wi

51、ll form in the drop.A Diffraction Experiment When the X-ray goes through the crystal, beams is diffracted and diffraction pattern is recorded on a detector. The crystal is rotated a certain degree while this pattern is recorded. A series of frames are collected. Determine the size of the unit cell b

52、y Braggs law: 2d sin = d= /(2* sin )./Xray/101index.htmlA Diffraction Experiment A narrow beam of x-rays (red) is taken out from the x-ray source through a collimating device. When the primary beam hits the crystal, most of it passes straight through, but some is diffract

53、ed by the crystal. These diffracted beams, which leave the crystal in many different directions, are recorded on a detector, either a piece of x-ray film or an area detector. The crystal was rotated one degree while this pattern was recorded. The pattern of RuBisCo was collected using polychromatic

54、radiation.Diffraction of X-rays by a Crystal(a) When a beam of x-rays (red) shines on a crystal all atoms in the crystal scatter x-rays in all directions. Most of these scattered x-rays cancel out, but in certain directions (blue arrow) they reinforce each other and add up to a diffracted beam. Diff

55、erent sets of parallel planes (b) can be arranged through the crystal so that each corner of all unit cells is on one of the planes of the set. X-ray diffraction can be regarded as reflection of the primary beam from sets of parallel planes in the crystal, separated by a distance d. The primary beam

56、 strikes the planes at an angle and the reflected beam leaves at the same angle, the reflection angle. X-rays (red) that are reflected from the lower plane have traveled farther than those from the upper plane by a distance BC + CD, which is equal to 2dsin.Reflection can only occur when this distanc

57、e is equal to the wavelength l of the x-ray beam and Braggs law (2d sin = l). To determine the size of the unit cell, the crystal is oriented in the beam so that reflection is obtained from the specific set of planes in which any two adjacent planes are separated by the length of one of the unit cel

58、l axes. This distance, d, is then equal to l/(2sin). The wavelength, l, of the beam is 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

59、 then reoriented, and the procedure is repeated for the other two axes of the unit cell. Diffraction of X-rays by a CrystalDiffraction of X-ray BeamsThe reflection angle, q, for a diffracted beam can be calculated from the distance (r) between the diffracted spot on a film and the position where the

60、 primary beam hits the film. From the geometry shown in the diagram, the tangent of the angle 2 = r/A. A is the distance between crystal and film that can be measured on the experimental equipment, while r can be measured on the film. Hence, can be calculated. The angle between the primary beam and the diff