Discrepancies between the NMR and X-ray Structures of Uncomplexed Barstar:  Analysis Suggests That Packing Densities of Protein Structures Determined by NMR Are Unreliable^,

Abstract

The crystal structure of the C82A mutant of barstar, the intracellular inhibitor of the Bacillus amyloliquefaciens ribonuclease barnase, has been solved to a resolution of 2.8 Å. The molecule crystallizes in the space group I4 1 with a dimer in the asymmetric unit. An identical barstar dimer is also found in the crystal structure of the barnase-barstar complex. This structure of uncomplexed barstar is compared to the structure of barstar bound to barnase and also to the structure of barstar solved using NMR. The free structure is similar to the bound state, and there are no significant main-chain differences in the 27-44 region involved in barstar binding to barnase. The C82A structure shows significant differences from the average NMR structure, both overall and in the binding region. In contrast to the crystal structure, the NMR structure shows an unusually high packing value based on the occluded surface algorithm, indicating errors in the packing of the structure. We show that the NMR structures of homologous proteins generally show large differences in packing value, while the crystal structures of such proteins have very similar packing values, suggesting that protein packing density is not well determined by NMR.

X‐ray crystallographic studies of the denaturation of ribonuclease S

“…Parameters such as accessibility, 51 and the OS packing value 52 were also examined for differences between the various structures. The mean protein packing value for individual residues as well as the mean packing value for each structure 53,54 were examined to quantitate changes in packing in the presence of urea and at low pH. Hydrogen bonds for the urea-soaked and low pH structures were compared to the control structures.…”

Calculation of Proteins’ Total Side-Chain Torsional Entropy and Its Influence on Protein–Ligand Interactions

“…All other cases include this dihedral potential together with subsets of side-chain interactions: S indicates steric energetics due to the repulsive part of the LJ potential; LJ indicates the full Lennard-Jones potential; IS indicates the implicit solvent; and HBSB indicates the hydrogen bonding and SB interactions. The proteins studied here are barstar (1a1945 ), calmodulin (3cln 46 ), crambin (1cbn 47 ), eglin c (1cse 48 ), GB3 (1igd 49 ), protein L (1hz6 50 ), PYP (1f9i 51 ), PZD2 (1r6j 52 ), SH2 (1d1z 53 ), CspA (1mjc 54 ), ubiquitin (1ubq 55 ), and tenascin (1ten 56 ). These results were calculated using Metropolis MC.…”

SecB Binds Only to a Late Native-like Intermediate in the Folding Pathway of Barstar and Not to the Unfolded State

“…The folding pathway of barstar has been extensively characterized using a variety of techniques (11)(12)(13). The folding, thermodynamics, and kinetics of several barstar mutants have also been studied (14)(15)(16), and crystal structures of both the free and bound protein are available (17,18). Of the two Pro residues in barstar, Pro48 occurs in the cis conformation in the folded state.…”