The microscopic interactions and mechanisms leading to nascent protein folding events are generally unknown. While such short time-scale events are difficult to study experimentally, molecular dynamics simulations of peptides can provide a useful model for studying events related to protein folding initiation. Recently, two extremely long molecular dynamics simulations (2.2 ns each) were carried out on the pentapeptide Tyr-Pro-Gly-Asp-Val [Tobias, D. J., Mertz, J. E., & Brooks, C. L., III (1991) Biochemistry 30, 6054-6058] that forms stable reverse turns in solution. Tobias et al. examined folding events in this large system (approximately 30,000 conformations) using traditional methods of trajectory analysis. The shear magnitude of this problem prompted us to develop an automated approach, based on self-organizing neural nets, to extract the key features of the molecular dynamics trajectory. The neural net is used to perform conformational clustering, which reduces the complexity of a system while minimizing the loss of information. The conformations were grouped together using distances in dihedral angle space as a measure of conformational similarity. The resulting clusters represent "conformational states", and transitions between these states were examined to identify mechanisms of conformational change. Many conformational changes involved the rotation of only a single dihedral angle, but concerted angle changes were also found. Most of the conformational information in the 30,000 samples from the full trajectories was retained in the relatively few resultant clusters, providing a powerful tool for analysis of an expanding base of large molecular simulations.

The clinical efficacy of carbapenem antibiotics depends on their resistance to the hydrolytic action of β-lactamase enzymes. The structure of the class D β-lactamase OXA-1 as an acyl-complex with the carbapenem doripenem was determined to 1.4 Å resolution. Unlike most class A and class C carbapenem complexes, the acyl carbonyl oxygen in OXA-1/doripenem is bound in the oxyanion hole. Interestingly, no water molecules were observed in the vicinity of the acyl linkage, providing an explanation for why carbapenems inhibit OXA-1. The side-chain amine of K70 remains fully carboxylated in the acyl structure, and the resulting carbamate group hydrogen bonds to the alcohol of the 6α-hydroxyethyl moiety of doripenem. The carboxylate attached to the β-lactam ring of doripenem is stabilized by a salt-bridge to K212 and a hydrogen bond with T213, in lieu of the interaction with an arginine side-chain found in most other β-lactamase/β-lactam complexes (eg. R244 in the class A member TEM-1). This novel set of interactions with the carboxylate results in a major shift of the carbapenem's pyrroline ring compared to the structure of the same ring in meropenem bound to OXA-13. Additionally, bond angles of the pyrroline ring suggest that after acylation, doripenem adopts the Δ 1 tautomer. These findings provide important insights into the role that carbapenems may have in the inactivation process of class D β-lactamases.Carbapenems are broad spectrum β-lactam antibiotics that represent one of the last lines of defense against severe bacterial infections. Doripenem ( Figure 1A) was approved for use in the United States in 2007 with indications for urinary tract infections and complicated intraabdominal infections (1). Though a broad spectrum antibiotic, it has shown higher efficacy than imipenem and meropenem in troublesome Gram-negative species such as Pseudomonas aeruginosa and Acinetobacter baumannii (1).One of the defining features of carbapenems is the presence of a 6α-hydroxyethyl moiety in lieu of the more bulky 6β amide side-chains found in other β-lactam antibiotics. This unique structural element is thought to be responsible for resistance of these drugs to the hydrolytic action of some β-lactamases (2,3). Classes A, C and D β-lactamases use a serine-nucleophile based covalent catalysis strategy (4). Interestingly, carbapenems are able to bind in the active site of these enzymes and can even proceed to a covalent acyl-intermediate with the active site serine (2,3,5). In many of these β-lactamases however, the water-dependent deacylation process is impaired. The end result is that carbapenems serve as effective irreversible inhibitors of many β-lactamases.The interaction of carbapenems with β-lactamases has been studied extensively. X-ray crystallographic studies of imipenem and meropenem bound to class A (3,6-9), class C (2) and class D (5) enzymes have yielded a wealth of structural detail about how the acyl-intermediate interacts with active site elements. In the case of most class A and class C enzymes, acylatio...

Local determinants of 3,0-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 5 1 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3,0-helices and multipleturn conformations, had strong amino acid sequence patterns not evident among a-helices. Aspartate occurred over twice as frequently in the N-cap position of 310-helices as in the N-cap position of a-helices. Unlike a-helices, 310-helices had few C-termini ending in a left-handed a conformation; most 310 C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3,0-and a-helices were also apparent, producing amphipathic 310-helices. Local interactions that stabilize 3,0-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of a-helices have been postulated to proceed through reverse-turn and 310-helix intermediates, sequence differences between 310-and ahelices can also lend insight into factors influencing a-helix initiation and propagation. Keywords: a-helix; amino acid determinants; 310-helicesThe conformation of a segment of protein backbone is determined both by local interactions, i.e., interactions within the segment and with residues neighboring in sequence, and by interactions with the segment's tertiary environment. We have undertaken a series of studies of three-dimensional protein crystal structures, involving the systematic classification of local protein conformations and the analysis of correlations between amino acid sequence and local conformation classes (Karpen, 1991). Strong correlations between amino acid sequence and local conformation can point to side-chain interactions that stabilize or destabilize a particular conformation relative to other conformations. In this paper, we examine 310-helices and how their amino acid sequences differ from a-helices. The 310-helical structures are relatively common in proteins; 4% of all residues in our select data base of 51 high-resolution proteins are involved in 310-helices. These short helices usually occur on the surface of a pro-

An efficient algorithm was characterized that determines the similarity in main chain conformation between short protein substructures. The algorithm computes Δt, the root mean square difference in ϕ and ψ torsion angles over a small number of amino acids (typically 3–5). Using this algorithm, large number of protein substrates comparisons were feasible. The parameter Δt was sensitive to variations in local protein conformation, and it correlates with Δr, the root mean square deviation in atomic coordinates. Values for Δt were obtained that define similarity thresholds, which determine whether two substructure are considered structurally similar. To set a lower bound on the similarity threshold, we estimated the component of Δt due to measurement noise fromcomparisons of independently refined coordinates of the same protein. A sample distribution of Δt from nonhomologous protein comparisons identified an upper bound on the similarity threshold, one that refrains from incorporating large numbers of nonmatching comparisons large numbers of nonmatching comparisons. Unlike methods based on Cα atoms alone, Δt was sensitive to rotations in the peptide plane, shown to occur in several proteins. Comparisons of homologus proteins by Δt showed that the active site torsion angles are highly conserved. The Δt method was applied to the α‐chain of human hemoglobin, where it readily demonstrated the local differences in the structures of different ligation states.