Protein-Conformational Structure Determination

Protein-Conformational Structure Determination Quaternary Structure 1 Protein-Conformational Structure Determination Determination of Quaternary Structure Need the “native” molecular weight Gel filtration Ultracentrifugation Need the subunit stoichiometry and molecular weight SDS-PAGE MALDI MALDI-MS Example 1: Example 3: Example 2: Native MW = 200 kDa Native MW = 360 kDa Native MW = 300 kDa Subunit MW = 50 kDa Subunit MW = 75 kDa, 50 kDa Subunit MW = 80 kDa, 40 kDa Subunit stoichiometry = equal Subunit stoichiometry = (means with half the size, the smaller one would (75:50) is 2:3 have to have half intensity on gel) a4 (means with the same amounts of total mass per protein, the smaller one would have to have the same intensity on gel) a2b3 a3b3 Protein-Conformational Structure Determination Analytical Ultracentrifugation In analytical ultracentrifugation, analytes are separated on the basis of their sedimentation when they experience a centrifugal force ~1.4 g/cm3 (Separation is based on Mass, Shape, Density). Usually carried out at speeds around 60,000 rpm There is a force due to buoyancy Fb and a frictional force on the molecule Ff, (centripetal force) both which oppose its movement through the medium Fs (centrifugal or sedimentary force). 2 Protein-Conformational Structure Determination TWO Types: Analytical Ultracentrifugation – Sedimentation Velocity Analytical Ultracentrifugation – Sedimentation Equilibrium Or, described mathematically…Fs + Fb + Ff = Sed. Vel. (rate) Solvent density in g/mL Specific Volume (occupied by 1 g of solute (cm3/g)) M is number average molecular weight (mN). If all species are the same, then M = Mr sedimentation velocity angular velocity radius from center of spin M (1 -n r ) µs = 2 ºs Nf wr Sedimentation coefficient (units of sec) coefficient of friction (g/mole•sec) Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Velocity M (1 -n r ) µs = 2 ºs Nf wr Units of sedimentation are seconds: Sedimentation coefficient (units of sec) 1 Svedberg = 1S = 10-13 s 1 -n r is called the buoyancy factor – and since 1/n approximates r, then if the (Say, “Vee-bar-rho”) density of the molecule is close to that of the solution 1 -n r = 0 and the s = 0. Proteins are more dense than solution and 1 -n r 1 -n r < 0 and the molecule floats. > 0, and the molecule sediments. !⩟ If the molecule is less dense than solution then (lipids or fats in a blood sample) 3 Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Velocity The most basic type of ultracentrifugation experiment is to measure the rate at which the molecule moves away from the center of rotation What is actually measured is the movement of the boundary between dissolved molecule and ‘empty’ buffer Based on mass, shape, and density t1 t2 t3 t4 Note shape of curvesless steep over time because of diffusion Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Velocity M (1 -n r ) µs = 2 ºs Nf wr 4 Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Equilibrium In sedimentation equilibrium, an equilibrium is established between sedimentation away from the center of rotation and diffusion towards the center of rotation (spin at much lower speeds) so we get no boundary between solute and meniscus This is anything that measures concentration! Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Equilibrium 5 Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Equilibrium Determining Molecular Weight Can be described by equation: CA (r ) = CA,0es ( r 2 -r02 ) / 2 where s = M (1 -n r )w 2 RT This equation can be expressed in terms of M: CA 2RT ln ___________ _ M = _________ 2 – r 2) 2 C (r (1 - vr) w 0 0 Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Equilibrium CA 2kT ln ___________ _ M = _________ C0(r2 – r02) (1 - vr) w2 Thus, we can get M. At most radii, its migrating “heavier” due to association. As you dilute the protein, A2 ⇌ 2A equilibrium shifts to the right. A2 ⇌ 2A Kd = [A]2/[A2] But recall, this M= mN, and depends on the number average molecular weight. So, if there are species with different molecular weights, as in a dissociation equilibrium of an oligomeric protein, this M will be sensitive to that dissociation. 6 Protein-Conformational Structure Determination Analytical Ultracentrifugation – Sedimentation Equilibrium CA 2kT ln ___________ _ M = _________ 2 C0(r2 – r02) (1 - vr) w Thus, we can get M. At most radii, its migrating “heavier” due to association. As you dilute the protein, A2 ⇌ 2A equilibrium shifts to the right. A2 ⇌ 2A Kd = [A]2/[A2] But recall, this M= mN, and depends on the number average molecular weight. So, if there are species with different molecular weights, as in a dissociation equilibrium of an oligomeric protein, this M will be sensitive to that dissociation. Protein Characterization Tertiary Structure 7 Protein-Conformational Structure Determination Analysis of 3o structure* • X-ray crystallography • NMR 59 *and 4o structure Protein-Conformational Structure Determination X-ray Crystallography for Protein Structure Comparison to other methods: l = 1.54 Å 8 Protein-Conformational Structure Determination X-ray Crystallography for Protein Structure 61 Protein-Conformational Structure Determination X-ray Crystallography for Protein Structure Closest layers of lines from atoms furthest apart in unit cell Intensities due to interference and amplification of waves Depends of whether waves are in or out of phase Furthers layers of lines from atoms closest together in unit cell = resolution 9 Protein-Conformational Structure Determination X-ray Crystallography for Protein Structure Resolution Protein-Conformational Structure Determination 10 Protein-Conformational Structure Determination Protein-Conformational Structure Determination X-ray Crystallography for Protein Structure 11 Protein-Conformational Structure Determination Typical X-ray crystallography structure The electron density map from the x-ray diffraction data is shown as a blue cage, and the model of the protein that “fits” this density in modeled inside of it. Protein-Conformational Structure Determination X-ray Crystallography for Protein Structure Stereo View 12 Protein-Conformational Structure Determination NMR for Protein Structure Protein-Conformational Structure Determination NMR for Protein Structure Correlation 2D spectroscopy (COSY) J-coupling R1 F angle R2 These intensities change depending on f-angle Ca H H H H R2 R1 C=O Hz ~4-5 N R1 R2 C=O Hz ~1-2 Can also get f & y angles from secondary chemical shifts (observed from known random coil shifts)(TALOS & ANGLOR) 13 Protein-Conformational Structure Determination NMR for Protein Structure Correlation 2D spectroscopy (COSY) J-coupling R1 F angle R2 These intensities change depending on f-angle Ca H H H H R2 R1 N C=O Hz ~4-5 R1 R2 C=O Hz ~1-2 Can also get f & y angles from secondary chemical shifts (observed from known random coil shifts)(TALOS & ANGLOR) Protein-Conformational Structure Determination NMR for Protein Structure Nuclear-Overhauser Effect Spectroscopy (NOESY) 14 Protein-Conformational Structure Determination Typical NMR structure Blue = b-sheet, red = ahelix, green = loops without 2° structure. Blue = N-term, red = C-term “Rainbow coloring.” Protein-Conformational Structure Determination NMR Structure of a Protein 15 Protein Characterization: Structure Determination Typical NMR structure Notice there are many overlapping structures that all fit the NMR data. Where it is tight, you have higher resolution and where it is loose you have parts of the molecule that are more mobile Protein-Conformational Structure Determination Protein Characterization Tertiary Structure Compare/Contrast X-ray crystallography and NMR: 1) Crystal vs. solution structures the same; not significant crystal constraints 2) NMR not as high resolution 3) NMR better at predicting regions that are dynamic; X-ray uses “B-factors” or even does not show, i.e., “disordered” 4) X-ray cannot distinguish “rotomers” of Asn, Gln, Thr; NMR is unambiguous 5) X-ray much better at larger structures; NMR has assignment problem only good for up to 30-40 kDa 16 Protein Characterization Secondary Structure 79 Protein-Conformational Structure Determination 17 Protein-Conformational Structure Determination Ala-Ala (assuming hard spheres) Gly-Gly NAc-Ala-Ala-NH2 (calculation with molecular dynamics including solvent (water)) Protein-Conformational Structure Determination Ala-Ala (assuming hard spheres) Gly-Gly NAc-Ala-Ala-NH2 (calculation with molecular dynamics including solvent (water)) 18 Protein-Conformational Structure Determination Ramachandran Plot Pyruvate kinase Protein-Conformational Structure Determination Secondary Structure 19 Protein-Conformational Structure Determination Secondary Structure Collagen 87 20 Protein Structure – Collagen Most abundant protein in mammals Extracellular: cartilage, tendons, bones, teeth, skin, vessels, lungs 3000 Å 88 Protein Structure – Collagen Primary & Secondary structure Primary structure Amino Acid composition Secondary structure Ramachandran plot f = -65° y = +130° CD of collagen Amino Acid Sequence (Gly-Pro/Ala-X)330 random coil X=hydroxyl-Pro, Glx, Arg, Asx, hydroxyl-Lys (Cd), Ser Hydroxylation reactions catalyzed by proline- and lysine-hydroxylase; require vitamin C 21 Protein Structure – Collagen Protein Structure – Collagen The 4 S’s for Collagen: Size Shape Stability Solubility 22 Protein Structure – Collagen F y Residues/ Repeat (Distance/repeat) 1.5 3.6 5.4 5.0 +135 3.4 2 6.8 - -119 +113 3.2 2 6.4 - 4 0 - i+1 -60 -30 - i+2 -90 0 4 0 - -60 120 - 3 9 14(triple) Structure Rise (°) (°) (Dist/residue) (Å) a-helix -57 -47 Anti- ⇌ b-sheet -139 Parallel ⥤ b-sheet b-turn-Type I b-turn-Type II i+1 i+2 80 0 - Collagen -65 +130 3 Pitch (Å) Diameter (Å) Size MW = 285,000 Da Long strands (3000 x 14 Å) helix dimensions/parameters Protein Structure – Collagen The Collagen Triple Helix C N Shape; Collagen-triple helix left handed right handed twist to three helices role of glycine Allows for packing of triple helix role of proline Locks the helix 23 Protein Structure – Collagen The Collagen Triple Helix C N Shape; Collagen-triple helix left handed right handed twist to three helices role of glycine Allows for packing of triple helix role of proline Locks the helix Protein Structure – Collagen Stability Packing of Gly Inter-stand H-bonds OH-P P G Inter-chain H-bonds 24 Protein Structure – Collagen Stability Melting (viscosity or CD vs. temp) cooperativity = sigmoidal plot role of hydroxyl-proline- Tm Melting of Collagen Species Body Temperature Calf 37 Shark, barracuda 26 Cod, deep sea redfish 14 Collagen Tm 39 29 16 50% Higher Tm correlates with higher OH-Pro/Pro Tm=39° Protein Structure – Collagen Pro Pro-OH Gly (Gly-Pro/Ala-X)330 (Gly-Pro-Prohydroxy) X=hydroxyl-Pro, Glx, Arg, Asx, hydroxyl-Lys (Cd), Ser 25 Protein Structure – Collagen The 4 S’s for Collagen: ✓ ✓ ✓ Size Shape Stability Solubility - not Protein Structure – Collagen Modified Residues in Collagen & Elastin 1.4-9% 0.5-3% 26 Protein Structure – Collagen Prolylhydroxylase ox red Lysylhydroxylase (uses a similar mechnism) Scurvy: Vitamin C Deficiency Protein Structure – Collagen Cross-linking of Collagen Solubility: insoluble due to cross-linking between FOUR triple helices using 3 Lys and 1 His residues lysyl oxidase à allysine LTQ cofactor merodesmosine 27 Protein Structure – Collagen Cross-linking of Collagen Solubility: insoluble due to cross-linking between FOUR triple helices using 3 Lys and 1 His residues lysyl oxidase à allysine LTQ cofactor merodesmosine Protein Structure – Collagen Cross-linking of Collagen NH3 NH3 Allysine aldol Modified from Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg: Sponk (talk)translated by Sponk (talk) - Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=11183362 28 Protein Structure – Collagen Cross-linking of Collagen NH3 NH3 Allysine aldol Modified from Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg: Sponk (talk)translated by Sponk (talk) - Tropokollagen_Quervernetzung_Lysyl_Oxidase_(DE).svg, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=11183362 Protein Structure – Collagen The 4 S’s for Collagen: ✓ ✓ ✓ ✓ Size: Long strands (3000 x 14 Å; ~300 Kda) Shape: left-handed triple helix Stability: controlled by degree of Pro-OH Solubility: - not How and where is collagen produced? biosynthesis 29 Protein Structure – Collagen 1) 2) 3) 4) 5) 6) 7) Transcription & translation Protein modification – hydroxylation – glycosylation quaternary structure & disulfides Secretion Procollagen processing Cross-linking Deposition Collagen Biosynthesis 108 Protein Structure – Collagen Collagen Disorders One thousand mutations have been identified in twelve out of more than twenty types of collagen. These mutations can lead to various diseases at the tissue level Diseases Scurvy – caused by lack of Vitamin C needed for hydroxylating enzyme that makes Pro-OH (Hyp) & Lys-OH (Hyl) Osteogenesis imperfecta – caused by a mutation in type 1 collagen weak bones and irregular connective tissue Ehlers-Danlos Syndrome – caused by a mutation in type 3 collagen (EDS, type 4) Ten different types of this disorder that lead to deformities in connective tissue. Osteoporosis – Not inherited genetically, brought on with age reduced levels of collagen in the skin and bone Knobloch syndrome – Caused by a mutation in the collagen XVIII gene protrusion of the brain tissue and degeneration of the retina 30