The role of London forces in defining noncentrosymmetric order of high dipole moment–high hyperpolarizability chromophores in electrically poled polymeric thin films

Graphs of second harmonic generation coefficients and electro-optic coefficients (measured by ellipsometry, attenuated total reflection, and two-slit interference modulation) as a function of chromophore number density (chromophore loading) are experimentally observed to exhibit maxima for polymers containing chromophores characterized by large dipole moments and polarizabilities. Modified London theory is used to demonstrated that this behavior can be attributed to the competition of chromophore-applied electric field and chromophore–chromophore electrostatic interactions. The comparison of theoretical and experimental data explains why the promise of exceptional macroscopic second-order optical nonlinearity predicted for organic materials has not been realized and suggests routes for circumventing current limitations to large optical nonlinearity. The results also suggest extensions of measurement and theoretical methods to achieve an improved understanding of intermolecular interactions in condensed phase materials including materials prepared by sequential synthesis and block copolymer methods.

Structural basis of DNA bending and oriented heterodimer binding by the basic leucine zipper domains of Fos and Jun

Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos–Jun–AP-1 complex may contribute to its functional versatility at different promoters.

Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase/reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II−. Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5,6-shift is 2.5 kcal/mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by ≈6 kcal/mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of ≈0.6 kcal/mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II− requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase.

pH gating of ROMK (Kir1.1) channels: Control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome

Inward-rectifier K+ channels of the ROMK (Kir1.1) subtype are responsible for K+ secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other Kir channels. Such anomalous titration of this lysine residue (Lys-80 in Kir1.1) is accomplished by the tertiary structure of the Kir protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in Kir1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pKa into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pKa of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of Kir channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

A streptavidin mutant with altered ligand-binding specificity

The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (Ka) of 1 × 106 M−1, two orders of magnitude higher than that for biotin (1 × 104 M−1). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 × 104 M−1) was lower than predicted (2.9 × 105 M−1), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.

Design of fast enzymes by optimizing interaction potential in active site

The diffusional encounter between substrate and enzyme, and hence catalytic efficiency, can be enhanced by mutating charged residues on the surface of the enzyme. In this paper we present a simple method for screening such mutations. This is based on our earlier result that electrostatic enhancement of the enzyme-substrate binding rate constant can be accounted for just by the interaction potential within the active site. Assuming that catalytic and structural integrity is maintained, the catalytic efficiency can be optimized by surface charge mutations which lead to stronger interaction potential within the active site. Application of the screening method on superoxide dismutase shows that only charge mutations close to the active site will have practical effect on the catalytic efficiency. This rationalizes a large number of findings obtained in previous simulation and experimental studies.

NMR structure of the bovine prion protein

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23–230), and a C-terminal fragment, bPrP(121–230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228–230, and an N-terminal flexibly disordered “tail” comprising 108 residues for the intact protein and 4 residues for bPrP(121–230), respectively. The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–226, and a short antiparallel β-sheet comprising the residues 128–131 and 161–164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166–172 leading from the β-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the “healthy” PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle.

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 2.1-Å resolution and XO at 2.5-Å resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form. Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion. Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433). This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme.

Energetics of the interaction between water and the helical peptide group and its role in determining helix propensities

The alanine helix provides a model system for studying the energetics of interaction between water and the helical peptide group, a possible major factor in the energetics of protein folding. Helix formation is enthalpy-driven (−1.0 kcal/mol per residue). Experimental transfer data (vapor phase to aqueous) for amides give the enthalpy of interaction with water of the amide group as ≈−11.5 kcal/mol. The enthalpy of the helical peptide hydrogen bond, computed for the gas phase by quantum mechanics, is −4.9 kcal/mol. These numbers give an enthalpy deficit for helix formation of −7.6 kcal/mol. To study this problem, we calculate the electrostatic solvation free energy (ESF) of the peptide groups in the helical and β-strand conformations, by using the delphi program and parse parameter set. Experimental data show that the ESF values of amides are almost entirely enthalpic. Two key results are: in the β-strand conformation, the ESF value of an interior alanine peptide group is −7.9 kcal/mol, substantially less than that of N-methylacetamide (−12.2 kcal/mol), and the helical peptide group is solvated with an ESF of −2.5 kcal/mol. These results reduce the enthalpy deficit to −1.5 kcal/mol, and desolvation of peptide groups through partial burial in the random coil may account for the remainder. Mutant peptides in the helical conformation show ESF differences among nonpolar amino acids that are comparable to observed helix propensity differences, but the ESF differences in the random coil conformation still must be subtracted.

Role of the J-domain in the cooperation of Hsp40 with Hsp70

The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes. Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain. The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2–75. Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2–75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2–388. NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP). Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2–75 respond to ATP-dependent changes in DnaK. The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions. The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Electric field-induced reorganization of two-component supported bilayer membranes

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory’s approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi–Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.

Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll–protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to ≈60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron–nuclear–nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from ≈2:1 to ≈5:1 in the mutants compared with ≈2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation–reduction midpoint potential.

Sodium channel selectivity filter regulates antiarrhythmic drug binding

Local anesthetic antiarrhythmic drugs block Na+ channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel’s selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na+ channel (μ1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na+ channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.

Solution structure of the antiapoptotic protein bcl-2

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-xL chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-xL. These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 α-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-xL. Comparison of the Bcl-2 structures to that of Bcl-xL shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.