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A spectroscopic investigation of the time course and products of NifS-mediated cluster assembly on wild-type NifU, wild-type and D37A NifU-1 and wild-type NifU-2 plus C-terminal domain fragments is reported. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.

Results demonstrate the ability of Isc homologue (sup)Nif IscA of Azotobacter vinelandii to bind iron and assemble iron-sulfur clusters through a NifS-directed pathway. Data indicate that (sup)Nif IscA lends itself as an alternative scaffold to NifU for the NifS-directed sequential assembly of iron-sulfur clusters.

A plant- and mammalian-type [2Fe-2S] ferredoxin, called ferredoxin 5 when expressed in Escherichia coli , is thermostable and differs from the iron-sulfur cluster ferredoxins by exhibiting deletions at N- and C-termini. Spectral analysis indicate that ferredoxin 5 and the Isc-Fd share structural similarities and redox potential properties.

The mechanism of IscS-directed Fe-S cluster assembly on the IscU scaffold is described using mass spectroscopy. The direct transfer of sulfane sulfur, S(super 0), from the cysteine persulfide on IscS to the cysteine residues on IscU indicates that this reaction constitutes the first step in Fe-S cluster biosynthesis on an IscU scaffold.

Nuclear magnetic resonance (NMR) and ultraviolet (UV) difference spectroscopy were used to assess the role of a number of tryptophan residues in the granular starch binding domain (SBD) of glucoamylase 1 from Aspergillus niger. Wild-type SBD and three variant (W563K, W590K, and W615K) proteins were produced using an A. niger expression system. Titration studies were conducted with beta-cyclodextrin (betaCD), a cyclic analogue of starch, as the ligand. The NMR studies show that the W563K and W590K variants only bind 1 equiv while the wild-type protein forms a 2:1 (ligand:protein) complex. It also clearly demonstrates the abolition of binding at site 1 and site 2 in W590K and W563K, respectively. UV difference spectroscopy was used to calculate dissociation constants with addition of betaCD: 14.4 microM (apparent) for the wild type, 28.0 microM for W563K, and 6.4 microM for W590K. The implication of this is that the two binding sites have unequal contributions to the overall binding of the SBD which may be related to functional differences between the two binding sites. The low stability of the third variant, W615K, suggests that this tryptophan is not involved in binding but has an essential structural role.

15N-labeled hen lysozyme has been studied by 2D and 3D NMR in order to characterize its dynamic behavior. The resonances of all main-chain amide nitrogen atoms were assigned, as were resonances of nitrogen atoms in 28 side chains. Relaxation measurements for the main-chain and arginine and tryptophan side-chain 15N nuclei used standard methods, and those for the 15N nuclei of asparagine and glutamine side chains used pulse sequences designed to remove unwanted relaxation pathways in the NH2 groups. The calculated order parameters (S2) show that the majority of main-chain amides undergo only small amplitude librational motions on a fast time scale (S2 > or = 0.8). Increased main-chain motion (0.5 < S2 < 0.8) is observed for a total of 19 residues located at the C-terminus, in loop and turn regions, and in the first strand of the main beta-sheet. Order parameters derived for the side chains range from 0.05 to 0.9; five of the six tryptophan residues have high order parameters (S2 > or = 0.8), consistent with their location in the closely packed core of the protein, whereas the order parameters between 0.05 and 0.3 for arginine residues confirm increased side-chain mobility at the protein surface. Order parameters for the side chains of asparagine and glutamine residues range from 0.2 to 0.8; high values are found for side chains that have low solvent accessible surfaces and well-defined chi 1 values, as measured by 3J alpha beta coupling constants. Many of the main-chain and side-chain groups with low order parameters have higher than average temperature factors in X-ray crystal structures and increased positional uncertainty in NMR solution structures. They also tend to lack persistent hydrogen bond interactions and protection against amide hydrogen exchange. The most significant correlations are found between residues with low order parameters and high surface accessibility in both crystal and solution structures. The results suggest that a lack of van der Waals contacts is a major determinant of side-chain and main-chain mobility in proteins.