We combine molecular dynamics simulations and new high-field NMR experiments to describe the solution structure of the Aβ [21][22][23][24][25][26][27][28][29][30] peptide fragment that may be relevant for understanding structural mechanisms related to Alzheimer's disease. By using two different empirical force-field combinations, we provide predictions of the three-bond scalar coupling constants ( 3 J H N H α), chemical-shift values, 13 C relaxation parameters, and rotating-frame nuclear Overhauser effect spectroscopy (ROESY) crosspeaks that can then be compared directly to the same observables measured in the corresponding NMR experiment of Aβ [21][22][23][24][25][26][27][28][29][30] . We find robust prediction of the 13 C relaxation parameters and medium-range ROESY crosspeaks by using new generation TIP4P-Ew water and Amber ff99SB protein force fields, in which the NMR validates that the simulation yields both a structurally and dynamically correct ensemble over the entire Aβ 21-30 peptide. Analysis of the simulated ensemble shows that all medium-range ROE restraints are not satisfied simultaneously and demonstrates the structural diversity of the Aβ 21-30 conformations more completely than when determined from the experimental medium-range ROE restraints alone. We find that the structural ensemble of the Aβ 21-30 peptide involves a majority population (~60%) of unstructured conformers, lacking any secondary structure or persistent hydrogen-bonding networks. However, the remaining minority population contains a substantial percentage of conformers with a β-turn centered at Val24 and Gly25, as well as evidence of the Asp23 to Lys28 salt bridge important to the fibril structure. This study sets the stage for robust theoretical work on Aβ 1-40 and Aβ 1-42 , for which collection of detailed NMR data on the monomer will be more challenging because of aggregation and fibril formation on experimental timescales at physiological conditions. In addition, we believe that the interplay of modern molecular simulation and high-quality NMR experiments has reached a fruitful stage for characterizing structural ensembles of disordered peptides and proteins in general.

Calmodulin (CaM) is the universal calcium sensor in eukaryotes, regulating the function of numerous proteins. Crystallography and NMR show that free CaM-4Ca2+ exists in an extended conformation with significant interdomain separation, but clamps down upon target peptides to form a highly compact structure. NMR has revealed substantial interdomain motions in CaM-4Ca2+, enabled by a flexible linker. In one instance, CaM-4Ca2+ has been crystallized in a compact configuration; however, no direct evidence for transient interdomain contacts has been observed in solution, and little is known about how large-scale interdomain motions contribute to biological function. Here we use paramagnetic relaxation enhancement (PRE) to characterize transient compact states of free CaM that are too sparsely populated to observe by traditional NMR methods. We show that unbound CaM samples a range of compact structures, populated at 5–10%, and that Ca2+ dramatically alters the distribution of these configurations in favor of states resembling the peptide-bound structure. In the absence of Ca2+, the target peptide binds only to the C-terminal domain, and the distribution of compact states is similar with and without peptide. These data suggest an alternative pathway of CaM action in which CaM remains associated with its kinase targets even in the resting state. Only CaM-4Ca2+, however, shows an innate propensity to form the physiologically active compact structures, suggesting that Ca2+ activates CaM not only through local structural changes within each domain but also through more global remodeling of interdomain interactions. Thus, these findings illustrate the subtle interplay between conformational selection and induced fit.

Transcription initiation by the sigma54 form of bacterial RNA polymerase requires hydrolysis of ATP by an enhancer binding protein (EBP). We present SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states. Structures of apo protein and that bound to AMPPNP or ADP-BeF(x) (ground-state mimics), ADP-AlF(x) (a transition-state mimic), or ADP (product) show substantial changes in the position of the GAFTGA loops that contact polymerase, particularly upon conversion from the apo state to the ADP-BeF(x) state, and from the ADP-AlF(x) state to the ADP state. Binding of the ATP analogs stabilizes the oligomeric form of the ATPase and its binding to sigma54, with ADP-AlF(x) having the largest effect. These data indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and sigma54, while subsequent hydrolysis and phosphate release drive the conformational change needed to open the polymerase/promoter complex.

Protein-protein association generally proceeds via the intermediary of a transient, lowly populated, encounter complex ensemble. The mechanism whereby the interacting molecules in this ensemble locate their final stereospecific structure is poorly understood. Further, a fundamental question is whether the encounter complex ensemble is an effectively homogeneous population of nonspecific complexes or whether it comprises a set of distinct structural and thermodynamic states. Here we use intermolecular paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to lowly populated states in the fast exchange regime, to characterize the mechanistic details of the transient encounter complex interactions between the N-terminal domain of Enzyme I (EIN) and the histidine-containing phosphocarrier protein (HPr), two major bacterial signaling proteins. Experiments were conducted at an ionic strength of 150 mM NaCl to eliminate any spurious nonspecific associations not relevant under physiological conditions. By monitoring the dependence of the intermolecular transverse PRE (Γ 2 ) rates measured on 15 N-labeled EIN on the concentration of paramagnetically labeled HPr, two distinct types of encounter complex configurations along the association pathway are identified and dissected. The first class, which is in equilibrium with and sterically occluded by the specific complex, probably involves rigid body rotations and small translations near or at the active site. In contrast, the second class of encounter complex configurations can coexist with the specific complex to form a ternary complex ensemble, which may help EIN compete with other HPr binding partners in vivo by increasing the effective local concentration of HPr even when the active site of EIN is occupied.enzyme I-histidine containing phosphocarrier protein complex | encounter complex | lowly populated states | NMR | phosphotransferase system S pecific protein-protein interactions underlie virtually every process in the cell. In general, specific protein-protein recognition proceeds via a two-step process (1-3): weak association via diffusion-controlled intermolecular collisions results in the formation of an ensemble of short-lived, encounter complexes located in multiple local free energy minima of a two-dimensional funnel-like energy landscape on the protein surface (4); subsequent rearrangement along the energy landscape, involving translations and rotations of the two partner proteins, permits the global free energy minimum to be located, resulting in the formation of a well-defined specific complex stabilized by a defined set of electrostatic and van der Waals interactions. From a functional perspective, encounter complex ensembles are thought to play a critical role in fine tuning reaction fluxes inside the cell (5), enhancing association on-rates by increasing the interaction crosssection and reducing the conformational search space on the path to the specific complex (6-11). Despite the importance of encounter complex ensembles in...

At high DNA concentration, as found in the nucleus, DNA-binding proteins search for specific binding sites by hopping between separate DNA strands. Here, we use 15 Nz-exchange transverse relaxation optimized NMR spectroscopy to characterize the mechanistic details of intermolecular hopping for the multidomain transcription factor, human Oct-1. Oct-1 is a member of the POU family of transcription factors and contains two helix-turn-helix DNA-binding domains, POU HD and POUS, connected by a relatively short flexible linker. The two domains were found to exchange between specific sites at significantly different rates. The cotranscription factor, Sox2, decreases the exchange rate and equilibrium dissociation constant for Oct-1 >5-fold and Ϸ20-fold, respectively, by slowing the exchange rate for the POU S domain. DNAdependent exchange rates measured at physiological ionic strength indicate that the two domains use both an intersegmental transfer mechanism, which does not involve the intermediary of free protein, and a fully dissociative or jumping mechanism to translocate between cognate sites. These data represent an example of dissecting domain-specific kinetics for protein-DNA association involving a multidomain protein and provide evidence that intersegmental transfer involves a ternary intermediate, or transition state in which the DNA-binding domains bridge two different DNA fragments simultaneously. intermolecular translocation ͉ protein-DNA interaction ͉ 15 N2-exchange NMR spectroscopy ͉ domain-specific kinetics ͉ target searching

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A genetic linkage map of grape was constructed, utilizing 116 progeny derived from a cross of two Vitis rupestris x V. arizonica interspecific hybrids, using the pseudo-testcross strategy. A total of 475 DNA markers-410 amplified fragment length polymorphism, 24 inter-simple sequence repeat, 32 random amplified polymorphic DNA, and nine simple sequence repeat markers-were used to construct the parental maps. Markers segregating 1:1 were used to construct parental framework maps with confidence levels >90% with the Plant Genome Research Initiative mapping program. In the maternal (D8909-15) map, 105 framework markers and 55 accessory markers were ordered in 17 linkage groups (756 cM). The paternal (F8909-17) map had 111 framework markers and 33 accessory markers ordered in 19 linkage groups (1,082 cM). One hundred eighty-one markers segregating 3:1 were used to connect the two parental maps' parents. This moderately dense map will be useful for the initial mapping of genes and/or QTL for resistance to the dagger nematode, Xiphinema index, and Xylella fastidiosa, the bacterial causal agent of Pierce's disease.