Chemical shifts and three-dimensional protein structures

Summary During the past three years it has become possible to compute ab initio the ¹³C, ¹⁵N and ¹⁹F NMR chemical shifts of many sites in native proteins. Chemical shifts are beginning to become a useful supplement to more established methods of solution structure determination, and may find utility in solid-state analysis as well. From ¹³C NMR, information on , and torsions can be obtained, permitting both assignment verification, and structure refinement and prediction. For ¹⁵N, both torsional and hydrogen-bonding effects are important, while for ¹⁹F, chemical shifts are primarily indicators of the local charge field. Chemical shift calculations are still slow, but shielding hypersurfaces — the shift as a function of the dihedral angles that define the molecular conformation — are becoming accessible. Over the next few years, theoretical and computer hardware improvements will enable more routine use of chemical shifts in structural studies, including the study of metal-ligand interactions, the analysis of drug and substrate binding and catalysis, the study of folding/unfolding pathways, as well as the characterization of conformational substates. Rather than simply being a necessary prerequisite for multidimensional NMR, chemical shifts and chemical shift non-equivalence due to folding are now beginning to be useful for structural characterization.     

Setting up and running molecular dynamics simulations of membrane proteins

Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins.     

Prediction of charge-induced molecular alignment: residual dipolar couplings at pH 3 and alignment in surfactant liquid crystalline phases

Recently we reported that the alignment tensor of a biological macromolecule, which was dissolved in a dilute suspension of highly negatively charged filamentous phage at close to neutral pH, can be predicted from the molecule’s 3D charge distribution and shape (Zweckstetter et al. 2004). Here it is demonstrated that this approach is also applicable to alignment of proteins in liquid crystalline phases formed by filamentous phage at low pH. Residual dipolar couplings (RDCs) predicted by our simple electrostatic model for the B1 domain of protein G in fd phage at pH 3 fit very well with the experimental values. The sign of charge–shape predicted one-bond ¹H–¹⁵N dipolar couplings for the B1 domain of protein G (GB1) was inverted at pH 3 compared to neutral pH, in agreement with experimental observations. Our predictions indicate that this is a feature specific for GB1. In addition, it is shown that RDCs induced in the protein ubiquitin by the presence of a positively charged surfactant system comprising cetylpyridinium bromide/hexanol/sodium bromide can be predicted accurately by a simple electrostatic alignment model. This shows that steric and electrostatic interactions dominate weak alignment of biomolecules for a wide range of pH values both in filamentous phage and in surfactant liquid crystalline phases.     

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.     

Electrostatic effects in short superhelical DNA

We present Monte Carlo simulations of the equilibrium configurations of short closed circular DNA that obeys a combined elastic, hard-sphere, and electrostatic energy potential. We employ a B-spline representation to model chain configuration and simulate the effects of salt on chain folding by varying the Debye screening parameter. We obtain global equilibrium configurations of closed circular DNA, with several imposed linking number differences, at two salt concentrations (specifically at the extremes of no added salt and the high salt regime), and for different chain lengths. Minimization of the composite elastic/long-range potential energy under the constraints of ring closure and fixed chain length is found to produce structures that are consistent with the configurations of short supercoiled DNA observed experimentally. The structures generated under the constraints of an electrostatic potential are less compact than those subjected only to an elastic term and a hard-sphere constraint. For a fixed linking number difference greater than a critical value, the interwound structures obtained under the condition of high salt are more compact than those obtained under the condition of no added salt. In the case of no added salt, the electrostatic energy plays a dominant role over the elastic energy in dictating the shape of the closed circular DNA. The DNA supercoil opens up with increasing chain length at low salt concentration. A branched three-leaf rose structure with a fixed linking number difference is higher in energy than the interwound form at both salt concentrations employed here.     

Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein

Histidine pK(a) values were measured in charge-reversal (K78E, K97E, K127E, and K97E/K127E) and charge-neutralization (E10A, E101A, and R35A) mutants of staphylococcal nuclease (SNase) by (1)H-NMR spectroscopy. Energies of interaction between pairs of charges (DeltaG(ij)) were obtained from the shifts in pK(a) values relative to wild-type values. The data describe the distance dependence and salt sensitivity of pairwise coulombic interactions. Calculations with a continuum electrostatics method captured the experimental DeltaG(ij) when static structures were used and when the protein interior was treated empirically with a dielectric constant of 20. The DeltaG(ij) when r(ij) < or = 10 A were exaggerated slightly in the calculations. Coulomb's law with a dielectric constant near 80 and a Debye-Hückel term to account for screening by the ionic strength reproduced the salt sensitivity and distance dependence of DeltaG(ij) as well as the structure-based method. In their interactions with each other, surface charges behave as if immersed in water; the Debye length describes realistically the distance where interactions become negligible at a given ionic strength. On average, charges separated by distances (r(ij)) approximately 5 A interacted with DeltaG(ij) approximately 0.6 kcal/mole in 0.01 M KCl, but DeltaG(ij) decayed to < or =0.10 kcal/mole when r(ij) = 20 A. In 0.10 M KCl, DeltaG(ij) approximately 0.10 kcal/mole when r(ij) = 10 A. In 1.5 M KCl, only short-range interactions with r(ij) < or = 5 A persisted. Although at physiological ionic strengths the interactions between charges separated by more than 10 A are extremely weak, in situations where charge imbalance exists many weak interactions can cumulatively produce substantial effects.     

Electrostatic Contributions to Colchicine Binding within Tubulin Isotypes

Tubulin, the structural subunit of microtubules, is the target of some highly successful anti-tumor drugs. Most of these drugs bind to the β-tubulin resulting in the inhibition of microtubule dynamics and eventually cell death. The varied cellular distribution of several human isotypes of β -tubulin provides a platform upon which to construct novel chemotherapeutic agents that are able to differentiate between these types of cells. To test this hypothesis, we have previously created homology models of the nine most frequently observed human β -tubulin isotypes and analyzed them for differences in the colchicine-binding site. Here, we describe the electrostatic properties of the colchicine binding site and how this may affect calculated drug binding affinities between the β -tubulin isotypes.     

Unraveling the Role of the C-terminal Helix Turn Helix of the Coat-binding Domain of Bacteriophage P22 Scaffolding Protein

Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the β-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the β-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction.     

An Extended Active-site Motif Controls the Reactivity of the Thioredoxin Fold

Proteins belonging to the thioredoxin (Trx) superfamily are abundant in all organisms. They share the same structural features, arranged in a seemingly simple fold, but they perform a multitude of functions in oxidative protein folding and electron transfer pathways. We use the C-terminal domain of the unique transmembrane reductant conductor DsbD as a model for an in-depth analysis of the factors controlling the reactivity of the Trx fold. We employ NMR spectroscopy, x-ray crystallography, mutagenesis, in vivo functional experiments applied to DsbD, and a comparative sequence analysis of Trx-fold proteins to determine the effect of residues in the vicinity of the active site on the ionization of the key nucleophilic cysteine of the -CXXC- motif. We show that the function and reactivity of Trx-fold proteins depend critically on the electrostatic features imposed by an extended active-site motif.