(φ,ψ)₂ motifs: a purely conformation-based fine-grained enumeration of protein parts at the two-residue level

A deep understanding of protein structure benefits from the use of a variety of classification strategies that enhance our ability to effectively describe local patterns of conformation. Here, we use a clustering algorithm to analyze 76,533 all-trans segments from protein structures solved at 1.2 Å resolution or better to create a purely φ,ψ-based comprehensive empirical categorization of common conformations adopted by two adjacent φ,ψ pairs (i.e., (φ,ψ)₂ motifs). The clustering algorithm works in an origin-shifted four-dimensional space based on the two φ,ψ pairs to yield a parameter-dependent list of (φ,ψ)₂ motifs, in order of their prominence. The results are remarkably distinct from and complementary to the standard hydrogen-bond-centered view of secondary structure. New insights include an unprecedented level of precision in describing the φ,ψ angles of both previously known and novel motifs, ordering of these motifs by their population density, a data-driven recommendation that the standard C^α_i…C^α_i + 3 < 7 Å criteria for defining turns be changed to 6.5 Å, identification of β-strand and turn capping motifs, and identification of conformational capping by residues in polypeptide II conformation. We further document that the conformational preferences of a residue are substantially influenced by the conformation of its neighbors, and we suggest that accounting for these dependencies will improve protein modeling accuracy. Although the CUEVAS-4D(r₁₀?₁₄) ‘parts list’ presented here is only an initial exploration of the complex (φ,ψ)₂ landscape of proteins, it shows that there is value to be had from this approach, and it opens the door to more in-depth characterizations at the (φ,ψ)₂ level and at higher dimensions.     

Structure of the Cdt1 C‐terminal domain: Conservation of the winged helix fold in replication licensing factors

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2‐7 complex onto the origin of chromosome. The C‐terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2‐7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X‐ray crystallography and solution NMR spectroscopy, respectively. While the N‐terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172–368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.     

Model quality assessment using distance constraints from alignments

Given a set of alternative models for a specific protein sequence, the model quality assessment (MQA) problem asks for an assignment of scores to each model in the set. A good MQA program assigns these scores such that they correlate well with real quality of the models, ideally scoring best that model which is closest to the true structure. In this article, we present a new approach for addressing the MQA problem. It is based on distance constraints extracted from alignments to templates of known structure, and is implemented in the Undertaker program for protein structure prediction. One novel feature is that we extract noncontact constraints as well as contact constraints. We describe how the distance constraint extraction is done and we show how they can be used to address the MQA problem. We have compared our method on CASP7 targets and the results show that our method is at least comparable with the best MQA methods that were assessed at CASP7. We also propose a new evaluation measure, Kendall's τ, that is more interpretable than conventional measures used for evaluating MQA methods (Pearson's r and Spearman's ρ). We show clear examples where Kendall's τ agrees much more with our intuition of a correct MQA, and we therefore propose that Kendall's τ be used for future CASP MQA assessments. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Evolutionary origin of a secondary structure: π-helices as cryptic but widespread insertional variations of α-helices that enhance protein functionality

Formally annotated π-helices are rare in protein structures but have been correlated with functional sites. Here, we analyze protein structures to show that π-helices are the same as structures known as α-bulges, α-aneurisms, π-bulges, and looping outs, and are evolutionarily derived by the insertion of a single residue into an α-helix. This newly discovered evolutionary origin explains both why π-helices are cryptic, being rarely annotated despite occurring in 15% of known proteins, and why they tend to be associated with function. An analysis of π-helices in the diverse ferritin-like superfamily illustrates their tendency to be conserved in protein families and identifies a putative π-helix-containing primordial precursor, a “missing link” intermediary form of the ribonucleotide reductase family, vestigial π-helices, and a novel function for π-helices that we term a “peristaltic-like shift.” This new understanding of π-helices paves the way for this generally overlooked motif to become a noteworthy feature that will aid in tracing the evolution of many protein families, guide investigations of protein and π-helix functionality, and contribute additional tools to the protein engineering toolkit.     

A rice glutamate receptor-like gene is critical for the division and survival of individual cells in the root apical meristem

Glu receptors are known to function as Glu-activated ion channels that mediate mostly excitatory neurotransmission in animals. Glu receptor-like genes have also been reported in higher plants, although their function is largely unknown. We have identified a rice (Oryza sativa) Glu receptor-like gene, designated GLR3.1, in which mutation by T-DNA insertion caused a short-root mutant phenotype. Histology and DNA synthesis analyses revealed that the mutant root meristematic activity is distorted and is accompanied by enhanced programmed cell death. Our results supply genetic evidence that a plant Glu receptor-like gene, rice GLR3.1, is essential for the maintenance of cell division and individual cell survival in the root apical meristem at the early seedling stage.     

The calorimetric criterion for a two-state process revisited.

The "calorimetric criterion" is one of the important experimental approaches for determining whether protein folding is an "all-or-none" two-state transition (i.e., whether intermediates are present at equilibrium). The calorimetric criterion states that the equivalence of the "measured" calorimetric enthalpy change and the effective two-state van't Hoff enthalpy change demonstrates that there is a two-state transition. This paper addresses the essential question of whether the calorimetric criterion is a necessary and sufficient condition for a two-state process and shows that it is necessary but not sufficient by means of specific examples. Analysis of simple models indicates that the heat capacity curve, regardless of whether it originates from a two-state process or not, can always be decomposed in such a way that the calorimetric criterion is satisfied. Exact results for a three-state model and a homopolymer tetramer demonstrate that the deviation from the calorimetric criterion is not simply related to the population of intermediate states. Analysis of a three-helix bundle protein model, which has a two-state folding from a random coil to ordered (molten) globule, shows that the calorimetric criterion may not be satisfied if the standard linear interpolation of baselines (weighted or unweighted) is employed. A specific example also suggests that the more recently introduced deconvolution method is not necessarily better than the simple calorimetric criterion for distinguishing a two-state transition from a three-state transition. Although the calorimetric criterion is not a sufficient condition for a two-state process, it is likely to continue to be of practical utility, particularly when its results are shown to be consistent with those from other experimental methods.     

MCSS functionality maps for a flexible protein

The Multiple Copy Simultaneous Search (MCSS) methodology for finding energetically favorable positions and orientations of small functional groups in a binding site is extended to include flexibility of the target. This makes possible the finding of novel minima not present in a fixed structure and so extends the diversity of inhibitors that can be constructed starting with the MCSS procedure. Quenched molecular dynamics is used to generate energetically favorable positions and orientations of the functional groups in the field of a flexible protein. The method is applied to the viral protein HIV-1 protease with methanol and methyl ammonium as a test case. If the protein is quenched with many copies of functional groups randomly distributed in the binding site, the resulting minima have ligand-protein interaction energies that are, on average, less favorable than those obtained with standard MCSS. This is a consequence of the renormalized potential function employed in the Locally Enhanced Sampling (LES) approximation. However, local optimizations of existing MCSS minima with a flexible protein results in lower energy minima in regions of the protein that are of particular interest. Their use in constructing a consensus protein model for ligand design is discussed.     

The fundamentals of protein folding: bringing together theory and experiment

Experimental and theoretical studies together are providing insights into the mechanism by which proteins fold. Our present knowledge of the essential aspects of the folding reaction is outlined and some approaches, both theoretical and experimental, that are being developed to obtain a more detailed understanding of this complex process are described.     

Dependence of DNA polymerase replication rate on external forces: a model based on molecular dynamics simulations

Molecular dynamics simulations are presented for a Thermus aquaticus (Taq) DNA polymerase I complex (consisting of the protein, the primer-template DNA strands, and the incoming nucleotide) subjected to external forces. The results obtained with a force applied to the DNA template strand provide insights into the effect of the tension on the activity of the enzyme. At forces below 30 pN a local model based on the parameters determined from the simulations, including the restricted motion of the DNA bases at the active site, yields a replication rate dependence on force in agreement with experiment. Simulations above 40 pN reveal large conformational changes in the enzyme-bound DNA that may have a role in the force-induced exonucleolysis observed experimentally.     

DNA polymorphism: a comparison of force fields for nucleic acids

The improvements of the force fields and the more accurate treatment of long-range interactions are providing more reliable molecular dynamics simulations of nucleic acids. The abilities of certain nucleic acid force fields to represent the structural and conformational properties of nucleic acids in solution are compared. The force fields are AMBER 4.1, BMS, CHARMM22, and CHARMM27; the comparison of the latter two is the primary focus of this paper. The performance of each force field is evaluated first on its ability to reproduce the B-DNA decamer d(CGATTAATCG)(2) in solution with simulations in which the long-range electrostatics were treated by the particle mesh Ewald method; the crystal structure determined by Quintana et al. (1992) is used as the starting point for all simulations. A detailed analysis of the structural and solvation properties shows how well the different force fields can reproduce sequence-specific features. The results are compared with data from experimental and previous theoretical studies.