HydrogenExchange-2010

HydrogenExchange-2010 - Intermediates Detection and...

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Intermediates Detection and Hydrogen Exchange • NMR methods for detecting intermediates and excited states • Amide exchange • Methods for detecting Amide Exchange • Application of Amide Exchange to proteins
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NMR Dynamic Experiments CPMG and PRE are sensitive to low-lying excited states with populations > 0.5% H/D exchange can detect high energy excited states with much lower population Mittermaier et al (2009) Trends Biochem. Sci. 34 , 601.
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Exchange Spectroscopy (EXSY) Mittermaier et al (2009) Trends Biochem. Sci. 34 , 601.
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CPMG Relaxation Dispersion Exchange between ground state and excited state is in the millisecond time scale Shape of the dispersion depend on: • populations of the two states • chemical shift difference • the rate of exchange A B k AB k BA k ex = k AB + k BA k BA > k AB Baldwin et al (2009) Nature Chem Biol 5 , 808.
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Relaxation Dispersion Boehr et al (2006) Science 313 , 1638.
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Differences in chemical shifts between ground and excited states ( Δω ) correlate with differences in peak positions between intermediates ( Δδ ), suggesting the excited states are similar to the conformation of the intermediates in the preceding or following step in the catalytic cycle. Boehr et al (2006) Science 313 , 1638. Relaxation Dispersion (Continue)
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Residual Dipolar Coupling (RDC) Mittermaier et al (2009) Trends Biochem. Sci. 34 , 601.
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Mittermaier et al (2009) Trends Biochem. Sci. 34 , 601. Spin Relaxation and Paramagnetic Relaxation Enhancement
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Grey: N-terminal domain Blue: C-terminal domain, apo Red: C-terminal domain, holo • PRE of holo form agree with X- ray structure • PRE of apo form is larger than X-ray structure • The apo form is in a transient close form (5%) Mittermaier et al (2009) Trends Biochem. Sci. 34 , 601. PRE Study of Maltose Binding Protein
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Intermediates Determined by H/D Exchange Englander (2005) PNAS 102 , 4741.
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Hydrogen Exchange Method Hydrogen exchange (HX) techniques is described for measuring the approximate exchange rates of the more labile amide protons in a macromolecule. The exchangeable amides in proteins are:
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Exchangeable Nucleotides
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Hydrogen-Exchange Chemistry Hx rate is catalyzed by OH - and H + k intrinsic k ex = k oH [OH - ] + k H [H + ] + k w All exchange rates are referenced to random coil polyAla at 0 C. HX rates are sensitive to pH,local chemical environment, solvent, sidechain type, neighboring amino acids and temperature k intrinsic for each amino acid is different A minimum ~ pH 3.5 > 1hr at pH 3 < 1ms at pH 10 pD = pH* + 0.4 Bai. And Englander. (1993) Proteins, 17, 75; Koide S. . and Wright PE J Biomol NMR. 1995 Nov;6(3):306-12 .
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Simulated Exchange Rates for Labile Protons of Polypeptides In H 2 O solution at 25 °C. Im stands for imidazole
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This note was uploaded on 11/07/2011 for the course CHEM 8853R taught by Professor Gelbaum during the Fall '11 term at Georgia Tech.

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HydrogenExchange-2010 - Intermediates Detection and...

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