The molecular structure of the DNA A‐tract dodecamer d(CGCAAATTTGCG) complexed with the drug Hoechst 33258 has been determined by X‐ray diffraction analysis. The Hoechst molecule binds in the DNA minor groove covering the sequence AATTT of the central A‐tract, with the piperazine group close to one of the GC regions. The drug molecule makes two three‐centered hydrogen bonds from the nitrogen atoms of the benzimidazole rings to the N3 and O2 atoms of the DNA bases. Although a high propeller twist is observed in the A‐tract, only one unsymmetrical three‐centered hydrogen bond is present in the DNA major groove. The structure is compared with other minor‐groove‐binding drug complexes and the influence of these drugs on DNA A‐tracts is discussed.

The role of mechanical force in cellular processes is increasingly revealed by single molecule experiments and simulations of force-induced transitions in proteins. How the applied force propagates within proteins determines their mechanical behavior yet remains largely unknown. We present a new method based on molecular dynamics simulations to disclose the distribution of strain in protein structures, here for the newly determined high-resolution crystal structure of I27, a titin immunoglobulin (IG) domain. We obtain a sparse, spatially connected, and highly anisotropic mechanical network. This allows us to detect load-bearing motifs composed of interstrand hydrogen bonds and hydrophobic core interactions, including parts distal to the site to which force was applied. The role of the force distribution pattern for mechanical stability is tested by in silico unfolding of I27 mutants. We then compare the observed force pattern to the sparse network of coevolved residues found in this family. We find a remarkable overlap, suggesting the force distribution to reflect constraints for the evolutionary design of mechanical resistance in the IG family. The force distribution analysis provides a molecular interpretation of coevolution and opens the road to the study of the mechanism of signal propagation in proteins in general.

We have designed de novo 13 divergent spectrin SH3 core sequences to determine their folding properties. Kinetic analysis of the variants with stability similar to that of the wild type protein shows accelerated unfolding and refolding rates compatible with a preferential stabilization of the transition state. This is most likely caused by conformational strain in the native state, as deletion of a methyl group (Ile-->Val) leads to deceleration in unfolding and increased stability (up to 2 kcal x mol(-1)). Several of these Ile-->Val mutants have negative phi(-U) values, indicating that some noncanonical phi(-U) values might result from conformational strain. Thus, producing a stable protein does not necessarily mean that the design process has been entirely successful. Strained interactions could have been introduced, and a reduction in the buried volume could result in a large increase in stability and a reduction in unfolding rates.