Proteins proton exchange rates

UV-VIS Spectra Reduction Potentials Active-Site Protonations Charge on Proteins Self-Exchange Rate Constants Electron Transfer Routes Comparison with Rubredoxin Summary References... 

Wagner, G., Wiithrich, K. (1979) Correlation between the amide proton exchange rates and the denaturation temperatures in globular proteins related to the basic pancreatic trypsin inhibitor. 7 Mol Biol, 130 (1), 31-37. 

Other examples of the dependence of proton exchange rates on the pA s and on the sum of the concentrations of bases present in the reaction mixture are described by Englander Kallenbach (1983). These authors apply the above rationale to the interpretation of exchange rates of solvent components with different bases in protein and nucleic acid molecules. 

Labelling with alone can be sufficient to overcome spectral overlap for proteins of up to 20 kDa and, for these proteins, virtually complete resolution can often be achieved for the backbone amide groups in 2D-iH-i HSQC experiments. These experiments are very robust and can be used to determine amide proton exchange rates or chemical shift temperature coefficients. For high protein concentrations H-i HSQC data sets can be acquired rapidly typically within 10 min for a 2 mM sample or 2-3 hr for a 0.2 mM protein sample. Consequently, this experiment has become the mainstay of NMR approaches to monitor the binding of ligands to i -labelled proteins through titration experiments. 

Even more information may be obtained by the. separate determination of A// and AS from differential scanning calorimetry, from the binding of 8-anilino-l-naphthalenesul-fonic acid, a fluorescent dye which preferentially binds to the molten globule state, and from proton exchange rates determined by NMR spectroscopy. Ultimately, all of these methods are required for a complete characterization of the protein. The final test remains the determination of the three-dimensional structure by X-ray crystallography or NMR. For a soluble, stable, folded protein, this should generally be possible. 

Amide proton temperature coefficients and hydrogen exchange rates can provide information about hydrogen-bonding interactions and solvent sequestration in unfolded or partly folded proteins (Dyson and Wright, 1991). Abnormally low temperature coefficients, relative to random coil values, are a clear indication of local structure and interactions. 

Mandelate racemase, another pertinent example, catalyzes the kinetically and thermodynamically unfavorable a-carbon proton abstraction. Bearne and Wolfenden measured deuterium incorporation rates into the a-posi-tion of mandelate and the rate of (i )-mandelate racemi-zation upon incubation at elevated temperatures. From an Arrhenius plot, they obtained a for racemization and deuterium exchange rate was estimated to be around 35 kcal/mol at 25°C under neutral conditions. The magnitude of the latter indicated mandelate racemase achieves the remarkable rate enhancement of 1.7 X 10, and a level of transition state affinity (K x = 2 X 10 M). These investigators also estimated the effective concentrations of the catalytic side chains in the native protein for Lys-166, the effective concentration was 622 M for His-297, they obtained a value 3 X 10 M and for Glu-317, the value was 3 X 10 M. The authors state that their observations are consistent with the idea that general acid-general base catalysis is efficient mode of catalysis when enzyme s structure is optimally complementary with their substrates in the transition-state. See Reference Reaction Catalytic Enhancement... 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

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