Abstract: 1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (H2NADH) has been investigated as a reduced coenzyme analog in the reaction between trans-4-N,N-dimethylaminocinnamaldehyde (I) (lambdamax 398 nm, epsilonmax 3.15 X 10-4 M-minus 1 cm-minus 1) and the horse liver alcohol dehydrogenase-NADH complex. These equilibrium binding and temperature-jump kinetic studies establish the following. (i) Substitution of H2NADH for NADH limits reaction to the reversible formation of a new chromophoric species, lambdamax 468 nm, epsilonmax 5.8 x 10-4 M-minus 1 cm-minus 1. This chromophore is demonstrated to be structurally analogous to the transient intermediate formed during the reaction of I with the enzyme-NADH complex [Dunn, M. F., and Hutchison, J. S. (1973), Biochemistry 12, 4882]. (ii) The process of intermediate formation with the enzyme-NADH complex is independent of pH over the range 6.13-10.54. Although studies were limited to the pH range 5.98-8.72, a similar pH independence appears to hold for the H2NADH system. (iii) Within the ternary complex, I is bound within van der Waal's contact distance of the coenzyme nicotinamide ring. (iv) Formation of the transient intermediate does not involve covalent modification of coenzyme. Based on these findings, we conclude that zinc ion has a Lewis acid function in facilitating the chemical activation of the aldehyde carbonyl for reduction, and that reduced coenzyme plays a noncovalent effector role in this substrate activating step.

Abstract: An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.

Abstract: The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.