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Department of Chemistry and Biochemistry University of Lethbridge Biochemistry 4200 I. Physical Biochemistry Protein Interactions Ultraviolet(UV)-Spectroscopy UV light is electromagnetic radiation with a wavelength between 10 – 400 nm. Wavelengths between 250 – 310 nm (near UV) are used to study proteins as they are primarily absorbed by aromatic amino acids (Phe (260 nm), Tyr (278 nm), Trp (292 nm)) within proteins. UV absorption by homogeneous protein samples depends on the number, type, conformation, and microenvironment of all its aromatic amino acids. Binding of a ligand to a protein can alter the conformation (and/or microenvironment) of one or more aromatic amino acids in the protein and give rise to an altered UV-absorption spectra. What is measured at 190-220 nm? 1 Ultraviolet(UV)-Spectroscopy Typical Experiment: Required solutions, etc.: 1. dilute solution of protein in buffer 2. a stock solution of concentrated ligand in the same buffer 3. stock solution of buffer 4. quartz cuvettes (Why?) How to continue? What do we need to meaure? Ultraviolet(UV)-Spectroscopy 1) Blank Place buffer in cuvette, measure absorbance at each wavelength between 250 – 310 nm. This is the absorption due to the buffer solution. Set value at each point to baseline. 2) Protein Spectra Place protein solution in cuvette and measure the absorbance at each wavelength. This is the absorbance due to the protein in your sample. 3) Ligand Spectra Place ligand solution in cuvette and measure the absorbance at each wavelength. This is the absorbance due to the ligand (in the absence of protein). Typically, small ligands have little or no UV absorbance in this region of the spectrum. 2 Ultraviolet(UV)-Spectroscopy 4) Protein-Ligand complex Spectra Place mixture of Protein-ligand complex in cuvette, measure absorbance at each wavelength. This is the combined absorbance of the protein and ligand in your sample. 5) Difference Spectra Mathematically, each point of the protein spectra and each point of the ligand spectra are subtracted from the equivalent point of the protein-ligand spectra. The result is the UV absorbance associated with the conformational changes in the protein due to ligand target binding. protein protein +ligand Ultraviolet(UV)-Spectroscopy Applications: Characterization of proteins (e.g. spectroscopic fingerprint) Determination of concentration (Aλ = ελ / c) Protein interactions Limitations: Ligand binding not always results UV changes No information on the nature of interaction Sample purity 3 CD Spectroscopy Circular dichroism (CD) refers to the difference in absorption between Right- and left-handed circular polarized light by a chiral sample. ∆ A = (AL – AR) / A CD Spectroscopy Circular dichroism (CD) refers to the difference in absorption between Right- and left-handed circular polarized light by a chiral sample. ∆ A = (AL – AR) / A Circular dichroism (∆ A) is typically converted to 2 -1 -1 mean molar ellipticity per residue ( Θmr : deg cm mol residue ) Circular polarized radiation is the result of two plane polarized beams that are normal to one another. The resulting rotational component is either clockwise (right-handed) or counter-clockwise (left-handed). 4 CD Spectroscopy Right-handed circular polarized Plane polarized light pertendicular to plane + phase shifted within plane Left-handed circular polarized Plane polarized light pertendicular to plane + phase shifted within plane CD Spectroscopy 5 CD Spectroscopy CD spectra are run in the far-UV region of about 190 – 250 nm where Peptide bonds absorb. The technique is particularly sensitive to secondary structures. CD can be used to estimate the secondary structural content of a protein. In general, the CD signal at 215 nm indicates the sheet content and The signal at 208 nm and 222 nm are used to calculated the helical content. CD Spectroscopy 6 CD Spectroscopy Example: 2 -1 -1 An all helix polypetide has an ellipticity of -38000 deg cm mol res at 222 nm An all random polypetode has an ellipticity of -12000 deg cm2 mol-1 res-1 at 222 nm An unknown protein has an ellipticity of -23000 deg cm2 mol-1 res-1 at 222 nm CD Spectroscopy (-23000 - -38000) / (-12000 - -38000) = 0.58 1.00 – 0.58 = 0.42 → 42 % helix 7 CD Spectroscopy Notes on the accuracy of the method (compared to known structures): helix sheet turn other 95-100% < 75% < 25% < 90 % Surface Plasmon Resonance Plasmon resonance refers to the loss of a small fraction of the total energy of incident radiation at specific angles of incidence. Polarized light striking a metal surface transfers energy to the delocalized electrons of the metal, reducing the intensity of the reflected light. 8 Surface Plasmon Resonance For thin metal film, the primary determinant of the intensity of the reflected light is the refractive index of the material coating it. The thin metal film (Au) is coated with immobilized sample. Solutions containing ligand are then passed across the immobilized sample. If binding occurs, the refractive index of the coated metal film is changed and both the intensity and angle of reflection are changed. Surface Plasmon Resonance Radiation used is typically in the near infra-red at 780 nm The plasmon resonance signal is proportional to the molecular mass of the ligand. 9 Surface Plasmon Resonance Allows to determine kinetic parameters: kon and koff Typical response curve: What is the problem here ? Analytical Ultracentrifugation Analytical ultracentrifugation utilizes Extremely high centrifugal fields e.g. 500,000 x g Experiments yield molecular mass information. Allows to asses oligomeric state of proteins in solution. 10 Analytical Ultracentrifugation Theory of the technique is straightforward 1- downward force in centrifugal field:, force down = m (1 – νp ρs) ω2 r νp = specivic volume ρs = density of solvent ω = rotaion (radians) 2 – force opposing sedimentation (frictiona) f = friction coefficient v = velocity forceopp = f v 3 – In the case of a sedimenting macromolecule f v < m (1 – νp ρs) ω2 r Analytical Ultracentrifugation 3 – In the case of a sedimenting macromolecule rearranged f v < m (1 – ν ρ ) ω2 r p s v ω2 r m < f v < ω2 r S (1 – νp ρs) Sedimentation coefficient Substitution of particle mass by molar mass (M) and Avogadro’s number: S < M Nf (1 – νp ρs) Measuring the rate of sedimentation allows to calculate S and than M 11 Analytical Ultracentrifugation Experimentally, the rate of sedimentation can be monitored by UV-Absorption. Fluorescence Resonance Energy Transfer Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. E Distance, Å The efficiency of FRET is dependent on the distance between the two dyes. FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity. 12 Fluorescence Resonance Energy Transfer Primary conditions for FRET Donor and acceptor molecules must be in close proximity (typically 10–100 Å). The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor. Donor and acceptor transition dipole orientations must be approximately parallel. Fluorescence Resonance Energy Transfer Förster Radius The distance at which energy transfer is 50% efficient is defined by the Förster radius (R0). The magnitude of Ro is dependent on the spectral properties of the donor and acceptor dyes. R0 = 44Å E Distance, Å 13 Fluorescence Resonance Energy Transfer Protein Dynamics: Arrangement of the Kirromycin Stalled EF-Tu on the Ribosome Donor P A 30Å EF-Tukirr 30Å 60Å 60Å P A Fluorescence 50S 30S Acceptor 1.1 0.9 0.7 + Phe-tRNAPhe Qsy8 0.5 0 2 4 6 Time, s 8 10 14