Valine activation of feeding in the sea anemone Boloceroides.
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Selective deuteration is a general solution to the resolution problem which limits the application of double resonance experiments to the assignment of the 1H NMR spectra of proteins. Spin-decoupling and NOE experiments have been carried out on Lactobacillus casei dihydrofolate reductase and on selectively deuterated derivatives of the enzyme containing either [gamma-2H6]Val or [alpha, delta 2, epsilon 1-2H3]His, [alpha, delta 1, delta 2, epsilon 1, epsilon 2, zeta-2H6]Phe, [alpha, delta 1, epsilon 3, zeta 2, zeta 3, eta 2-2H6]Trp and [alpha, epsilon 1, epsilon 2-2H3]Tyr. When combined with ring-current shift calculations based on the crystal structure of the enzyme, these experiments allow us to assign 1H resonances of Val 61, Val 115, Tyr 46 and Tyr 68.
An iron-sulfur metalloprotein containing the 5-12 and 35-50 residues of Desulfovibrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val-5 and Glu-50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV-VIS spectrum of the reduced synthetic protein is very similar to that of native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (+/- 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre-requisite for protein formation. The Val-->Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.
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