N-acetyl Tryptophanamide Research Articles

Reorientation motions of N-acetyl-tryptophan-amide (NATA dipeptide) in aqueous solution and with co-solvents: Molecular dynamics vs hydrodynamic model

We present a study of peptide reorientational dynamics in solution analyzed from the perspective of fluorescence anisotropy decay (FAD) experiments, and atomistic molecular dynamics (MD) and continuum hydrodynamics modeling. Earlier, FAD measurements and MD simulations of the model dipeptide N-acetyltryptophanamide (NATA) in explicit water and in aqueous solutions of urea, guanidinium chloride, and proline co-solvents identified excellent agreement of MD results with experimental data, indicating the presence of significant effects of peptide–solvent interactions, and the overall tumbling of the peptide could be well described by contributions from individual conformers, represented by dihedral-restrained MD. Here, we extend these studies by analyzing dynamic inhomogeneity in the solutions and by developing a hydrodynamic model (HM) of the conformer dynamics. The MD simulation data indicate the presence of markedly different dynamic microenvironments for the four studied solutions, with the average water reorientations being different in all systems, partly reflecting the bulk viscosities. Additionally, the water dynamics also exhibited a marked slowdown in the vicinity of the co-solvents, especially chloride and proline. To gain further insight, we applied the HM to predict rotational correlation times of tryptophan for the individual NATA conformers identified in MD. The hydrodynamic results were in very good agreement with MD simulations for the individual structures, showing that the HM model provides a realistic description of rotational diffusion for rigid peptide structures. Overall, our study generated new microscopic insights into the complex nature of the structure and dynamics of peptide solvation shells for systems containing water and denaturing and stabilizing co-solvents.

Probing coupled motions of peptides in solution with fluorescence anisotropy and molecular dynamics simulation

Abstract Here we present an atomically detailed study of local and global motions in two peptide systems, N-acetyl-tryptophanamide (NATA) and a pentapeptide WA3K (WK5) combining detailed spectroscopic measurements and comprehensive computer simulations. We have measured fluorescence lifetimes, fluorescence anisotropy decay (FAD), far-UV circular dichroism (CD) spectra as a function of temperature, and carried out molecular dynamics (MD) simulation with an improved water model (TIP4P/EW). Fluorescence lifetime analysis showed the presence of a heterogeneous population of conformations in both peptides and a quenched fluorescence in WK5. Far-UV CD measurements quantified structural populations in both peptides specifying alpha-helix and sheet for NATA and 310 helix and polyproline II for WK5 and their variation with temperature. FAD showed a bi-exponentiality in the reorientation dynamics, indicating the presence coupled motions in solution. The faster time constants, 6 ps for NATA, and 26 ps for WK5 are nearly temperature-independent and the slower components, 60 ps for NATA and 246 ps for WK5 at room temperature, vary with temperature. These faster and slower time constants are likely associated with the initiation and propagation steps of the reorientation dynamics. Results from MD simulation are in good agreement with the measured reorientation time and structural conformations. MD analysis showed that the measured faster time constants may be ascribed to delocalized molecular vibrations. Kinetic analysis found motions in the 1 ns in NATA and 10 ns in WK5. Both peptides exhibited markedly slower internal fluctuations compared to freely moving systems in the absence of overall translations and rotations, indicating a significant coupling between local and global dynamics. This study, focusing on simple model systems, has generated new insights into the dynamic behavior of peptides in solution.

Dynamic elements and kinetics: Most favorable conformations of peptides in solution with measurements and simulations.

Small peptides in solution adopt a specific morphology as they function. It is of fundamental interest to examine the structural properties of these small biomolecules in solution and observe how they transition from one conformation to another and form functional structures. In this study, we have examined the structural properties of a simple dipeptide and a five-residue peptide with the application of far-UV circular dichroism (CD) spectroscopy as a function of temperature, fluorescence anisotropy, and all-atom molecular dynamics simulation. Analysis of the temperature dependent CD spectra shows that the simplest dipeptide N-acetyl-tryptophan-amide (NATA) adopts helical, beta sheet, and random coil conformations. At room temperature, NATA is found to have 5% alpha-helical, 37% beta sheet, and 58% random coil conformations. To our knowledge, this type of structural content in a simplest dipeptide has not been observed earlier. The pentapeptide (WK5) is found to have four major secondary structural elements with 8% 310 helix, 14% poly-L-proline II, 8% beta sheet, and 14% turns. A 56% unordered structural population is also present for WK5. The presence of a significant population of 310 helix in a simple pentapeptide is rarely observed. Fluorescence anisotropy decay (FAD) measurements yielded reorientation times of 45 ps for NATA and 120 ps for WK5. The fluorescence anisotropy decay measurements reveal the size differences between the two peptides, NATA and WK5, with possible contributions from differences in shape, interactions with the environment, and conformational dynamics. All-atom molecular dynamics simulations were used to model the structures and motions of these two systems in solution. The predicted structures sampled by both peptides qualitatively agree with the experimental findings. Kinetic modeling with optimal dimensionality reduction suggests that the slowest dynamic processes in the dipeptide involve sidechain transitions occurring on a 1 ns timescale. The kinetics in the pentapeptide monitors the formation of a distorted helical structure from an extended conformation on a timescale of 10 ns. Modeling of the fluorescence anisotropy decay is found to be in good agreement with the measured data and correlates with the main contributions of the measured reorientation times to individual conformers, which we define as dynamic elements. In NATA, the FAD can be well represented as a sum of contributions from representative conformers. This is not the case in WK5, where our analysis suggests the existence of coupling between conformational dynamics and global tumbling. The current study involving detailed experimental measurements and atomically detailed modeling reveals the existence of specific secondary structural elements and novel dynamical features even in the simplest peptide systems.

Cis/Trans Isomerization of Unsaturated Fatty Acids in Polysorbate 80 During Light Exposure of a Monoclonal Antibody-Containing Formulation.

Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization.

Probing Selection Mechanism of the Most Favorable Conformation of a Dipeptide in Chaotropic and Kosmotropic Solution.

Chaotropes like urea and guanidinium chloride (GdmCl) tend to destabilize, and kosmotropes like proline tend to stabilize folded structures of peptides and proteins. Here, we combine fluorescence anisotropy decay measurements and molecular dynamics simulations to gain a microscopic understanding of the molecular mechanism for shifting conformational preferences in aqueous, GdmCl, urea, and proline solutions of a simple model dipeptide, N-acetyl-tryptophan-amide (NATA). Measured anisotropy decay of NATA as a function of temperature, pH, and cosolvent concentrations showed reorientations moderately slower in GdmCl and urea and substantially slower in proline compared to those of aqueous environment. A small change in pH significantly slows orientation time in water and GdmCl and less markedly in urea. Computationally, we use molecular dynamics with dihedral restraints to separately analyze the motions and interactions of the representative NATA conformers in the four different solvent environments. This novel analysis provides a dissection of the observed overall diffusion rates into contributions from individual dipeptide conformations. The variation of rotational diffusion rates with conformation are quite large. Population-weighted averaging or using properties of the major cluster reproduces the dynamical features of the full unrestrained dynamics. Additionally, we correlate the observable diffusion rates with microscopic features of conformer size, shape, and solvation. This analysis uncovered underlying differences in detailed atomistic behavior of the three cosolvents-urea, GdmCl, and proline. For both urea and the pure water system we find good agreement with hydrodynamic theory, with diffusion rates primarily correlated with conformer size and shape. In contrast, for GdmCl and proline solutions, the variation in conformer diffusion rates was mostly determined by specific interactions with the cosolvents. We also find preferences for different molecular shapes by the three cosolvents, with increased preferential solvation of smaller and more spherical conformers by urea and larger and more elongated conformers by GdmCl and proline. Additionally, our results provide a basis for a simple approximate model of the effects of pH lowering on dipeptide conformational equilibria. The translational diffusion rates of NATA are less sensitive to conformations, but variation with solvation strength is similar to rotational diffusion. Our results, combining experiment and simulation, show that we can identify the individual peptide conformers with definite microscopic properties of shape, size, and solvation, that are responsible for producing physical observables, such as translational and orientational diffusion in the complex solvent environments of denaturants and osmolytes.

Permeation of the three aromatic dipeptides through lipid bilayers: Experimental and computational study.

The time-resolved parallel artificial membrane permeability assay with fluorescence detection and comprehensive computer simulations are used to study the passive permeation of three aromatic dipeptides-N-acetyl-phenylalanineamide (NAFA), N-acetyltyrosineamide (NAYA), and N-acetyl-tryptophanamide (NATA) through a 1,2-dioleoyl-sn-glycero-3-phospocholine (DOPC) lipid bilayer. Measured permeation times and permeability coefficients show fastest translocation for NAFA, slowest for NAYA, and intermediate for NATA under physiological temperature and pH. Computationally, we perform umbrella sampling simulations to model the structure, dynamics, and interactions of the peptides as a function of z, the distance from lipid bilayer. The calculated profiles of the potential of mean force show two strong effects-preferential binding of each of the three peptides to the lipid interface and large free energy barriers in the membrane center. We use several approaches to calculate the position-dependent translational diffusion coefficients D(z), including one based on numerical solution the Smoluchowski equation. Surprisingly, computed D(z) values change very little with reaction coordinate and are also quite similar for the three peptides studied. In contrast, calculated values of sidechain rotational correlation times τrot(z) show extremely large changes with peptide membrane insertion-values become 100 times larger in the headgroup region and 10 times larger at interface and in membrane center, relative to solution. The peptides' conformational freedom becomes systematically more restricted as they enter the membrane, sampling α and β and C7eq basins in solution, α and C7eq at the interface, and C7eq only in the center. Residual waters of solvation remain around the peptides even in the membrane center. Overall, our study provides an improved microscopic understanding of passive peptide permeation through membranes, especially on the sensitivity of rotational diffusion to position relative to the bilayer.

Reorientation Motion and Preferential Interactions of a Peptide in Denaturants and Osmolyte

Fluorescence anisotropy decay measurements and all atom molecular dynamics simulations are used to characterize the orientational motion and preferential interaction of a peptide, N-acetyl-tryptophan-amide (NATA) containing two peptide bonds, in aqueous, urea, guanidinium chloride (GdmCl), and proline solution. Anisotropy decay measurements as a function of temperature and concentration showed moderate slowing of reorientations in urea and GdmCl and very strong slowing in proline solution, relative to water. These effects deviate significantly from simple proportionality of peptide tumbling time to solvent viscosity, leading to the investigation of microscopic preferential interaction behavior through molecular dynamics simulations. Examination of the interactions of denaturants and osmolyte with the peptide backbone uncovers the presence of strongest interaction with urea, intermediate with proline, and weakest with GdmCl. In contrast, the strongest preferential solvation of the peptide side chain is by the nonpolar part of the proline zwitterion, followed by urea, and GdmCl. Interestingly, the local density of urea around the side chain is higher, but the GdmCl distribution is more organized. Thus, the computed preferential solvation of the side chain by the denaturants and osmolyte can account for the trend in reorientation rates. Analysis of water structure and its dynamics uncovered underlying differences between urea, GdmCl, and proline. Urea exerted the smallest perturbation of water behavior. GdmCl had a larger effect on water, slowing kinetics and stabilizing interactions. Proline had the largest overall interactions, exhibiting a strong stabilizing effect on both water-water and water-peptide hydrogen bonds. The results for this elementary peptide system demonstrate significant differences in microscopic behavior of the examined solvent environments. For the commonly used denaturants, urea tends to form disorganized local aggregates around the peptide groups and has little influence on water, while GdmCl only forms specific interactions with the side chain and tends to destabilize water structure. The protective osmolyte proline has the strongest and most specific interactions with the tryptophan side chain, and also stabilizes both water-water and water-peptide hydrogen bonds. Our results strongly suggest protein or peptide denaturation triggered by urea occurs by direct interaction, whereas GdmCl interacts favorably with side chains and destabilizes peptide-water hydrogen bonds. The stabilization of biopolymers by an osmolyte such as proline is governed by favorable preferential interaction with the side chains and stabilization of water.

Modeling the solvation of nonpolar amino acids in guanidinium chloride solutions.

It is common to denature proteins by using high temperatures or by adding guanidinium chloride (GdmCl). However, the physical mechanism of denaturation is not well understood. Based on extensive experimental data, we developed a thermodynamic binding-polynomial model for the process of transferring nonpolar amino acids from water into GdmCl solutions, as a function of temperature and GdmCl concentration. To mimic nonpolar amino acids, we utilized the model compound, N-acetyl-tryptophanamide (NATA). We find that all nonpolar amino acids behave like NATA, with a scale factor linearly dependent on the surface area. Our model with three thermodynamic parameters fully captures the nonlinear dependencies on both the temperature and GdmCl concentration: binding the first guanidinium ion (Gdm(+)) to NATA has favorable entropy and unfavorable enthalpy of desolvation (ΔS = +11.7 cal/mol, ΔH = +3.9 kcal/mol), while cooperativity of binding a second Gdm(+) has a small contribution (K = 0.032 ± 0.003). This model may be useful for a better understanding of protein denaturation by temperature and GdmCl.

Complexation of calix[4]arene hydroxymethyl-phosphonic acid with tryptophan and N-acetyl-tryptophan amide

The Host-Guest complexation of calixarene hydroxymethylphosphonic acid with tryptophan and N-acetyltryptophan amide has been investigated by the RP HPLC method in H2O/MeCN (99/1) solution (column support Hypersil CN, UV-detector, λ = 254 nm). Adsorption of calixarene hydroxymethylphosphonic acid on the Hypersil CN surface has been studied. It has been found that hydroxymethylphosphonic acid is characterized by reversible sorption on the Hypersil CN surface. The binding constants (KA = 23000 M-1 and 39000 M-1 for tryptophan and N-acetyltryptophan amide, respectively) of the supramolecular complexes have been calculated from the ratio between the capacity factors k’ of the Guest and the calixarene hydroxymethylphosphonic acid Host concentration in the mobile phase. The Gibbs free energies of the tryptophan and N-acetyltryptophan amide complexes are -24.84 and -26.15 kJ/mol, respectively. The molecular modelling of calixarene hydroxymethylphosphonic acid and its complexes with tryptophan and N-acetyltryptophan amide (Hyper Chem, version 8, force field PM3) has indicated that the complexes are stabilized by hydrogen bonds, electrostatic, π-π, and solvatophobic interactions. The geometric parameters of the energy minimized calixarene macrocycle and its complexes with tryptophan and N-acetyltryptophan amide have been calculated. According to the calculations it has been shown that the Host-Guest complexation does not change the flattened-cone conformation of calixarene. Finally, the inverse correlation has been found between the KA values of the complexes and the Log P values of the guest molecules.

The kinetics of the reaction of nitrogen dioxide with iron(II)- and iron(III) cytochrome c

The reactions of NO2 with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23°C. NO2 oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×105 and (1.1±0.1)×106M−1s−1, respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×106M−1s−1 at pH 7.2. NO2 oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×107M−1s−1 at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO2 oxidizes the iron center directly—most probably via reaction at the solvent-accessible heme edge—whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO2 to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO2 will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.

The Role of Site-Specific Hydrogen Bonding Interactions in the Solvation Dynamics of N-Acetyltryptophanamide

Measurements of the ultrafast broadband UV fluorescence of N-acetyltryptophanamide (NATA) provide detailed information on its relaxation patterns in three different solvents: methanol (MeOH), water and acetonitrile (ACN). Several processes leading to excited state solvation and cooling are found to occur on different characteristic time scales and are thoroughly analyzed. Comparison between protic MeOH and aprotic ACN allows one to single out a 12 ps component in the former, which is attributed to the rearrangement of H-bonds existing between the protic solvent and excited NATA. This significantly stabilizes the excited state and provides the molecule with an efficient cooling mechanism. The corresponding dynamics in water are much faster (<1.5 ps). Comparison with static spectroscopic properties stresses the role of site-specific H-bonding in controlling the fluorescence quantum yield of NATA in protic solvents. These findings are consistent with existing models that describe tryptophan quenching as a result of charge transfer from the indole to the amide assisted by H-bonding at the carbonyl site.

Fast repair of protein radicals by urate

The repair of tryptophan and tyrosine radicals in proteins by urate was studied by pulse radiolysis. In chymotrypsin, urate repairs tryptophan radicals efficiently with a rate constant of 2.7×108M−1s−1, ca. 14 times higher than the rate constant derived for N-acetyltryptophan amide, 1.9×107M−1s−1. In contrast, no repair of tryptophan radicals was observed in pepsin, which indicates a rate constant smaller than 6×107M−1s−1. Urate repairs tyrosine radicals in pepsin with a rate constant of 3×108M−1s−1―ca. 12 times smaller than the rate constant reported for free tyrosine―but not in chymotrypsin, which implies an upper limit of 1×106M−1s−1 for the corresponding rate constant. Intra- and intermolecular electron transfer from tyrosine residues to tryptophan radicals is observed in both proteins, however, to different extents and with different rate constants. Urate inhibits electron transfer in chymotrypsin but not in pepsin. Our results suggest that urate repairs the first step on the long path to protein modification and prevents damage in vivo. It may prove to be a very important repair agent in tissue compartments where its concentration is higher than that of ascorbate. The product of such repair, the urate radical, can be reduced by ascorbate. Loss of ascorbate is then expected to be the net result, whereas urate is conserved.

Computational Predictions of Exponential and Non-Exponential Tryptophan Fluorescence Decay in NATA, the Villin Headpiece Subdomain, and other Proteins

Decay of fluorescence from a single Tryptophan (Trp) in a peptide or protein is sometimes exponential and sometimes non-exponential. Understanding the details of this behavior has proven elusive. We have had qualitative success predicting average fluorescence quantum yields for a variety of proteins based on the stabilization of the charge transfer (CT) state arising from electron transfer from the aromatic ring to a backbone amide by the protein electrostatic environment. Here we report 100ns - 1 microsecond MD simulations, augmented by quantum mechanical (ZINDO) computations of the fluorescing and CT states, on N-acetyl-tryptophan-amide (NATA) and 20 single Trp proteins, including the highly studied fast folding villin headpiece. Although the environment for NATA in water is very similar to that of solvent exposed Trps on the surface of a protein, which almost always exhibit non-exponential decay, NATA shows a surprisingly pure single-exponential decay. In our simulations, all possible rotamer states are well represented, and transitions between rotamers happen at a rate of about 1 per 5 ns. Preliminary results indicate that rare conformations in which an amide carbonyl is H-bonded by 2-3 waters produce spikes of high quenching, more or less independent of rotamer state. Survival curve averaging of the 600 ns trajectory yields a single exponential decay of near 3 ns. For folded villin at 300 K, we find that rotamer transitions on Trp occur only every ∼100 ns and that quenching by the nearby His+ happens only during these transient events, although the helix is always intact. Villin is therefore predicted to show extreme heterogeneity in lifetimes. This provides a mechanism for Trp fluorescence to report the global folding rate. Heterogeneity for the other proteins will also be discussed.

Solubilities and Transfer Free Energies of Hydrophobic Amino Acids in Polyol Solutions: Importance of the Hydrophobicity of Polyols

The purpose of this work was to investigate the difference in the hydrophobicities of various polyols and the nature of interactions between hydrophobic amino acid side chains and polyols. The interactions were explored by conducting solubility studies of three amino acid derivatives, N-acetyl tryptophanamide (NATA), N-acetyl leucinamide (NALA), and N-acetyl glycinamide (NAGA), in the solutions of sorbitol, sucrose, trehalose, glycerol, ethylene glycol, ribose, and deoxyribose. Hydrophobicity index of polyols was calculated using molecular modeling. An increase in the solubility of the hydrophobic side chains of tryptophan and leucine was observed with an increase in the hydrophobicity index of polyols. Transfer free energies of NATA from water to polyols solutions were negative, whereas those for NALA were positive for all polyols except glycol. This study shows that the hydrophobic nature of polyols plays an important role in polyol-side chain interactions. Solubility behavior observed for NATA and NALA in different polyols indicates that polyols can interact differently with the same side chain depending on the nature of the polyol and the side chain.

Relationship between the wavelength maximum of a protein and the temperature dependence of its intrinsic tryptophan fluorescence intensity

Proper determination of the temperature dependence of intrinsic tryptophan fluorescence intensity in native and denatured states is an essential prerequisite for extracting the free energy of protein unfolding from the thermal denaturation profile. The most common method employed in determining the temperature dependence of these conformations is through the determination of slopes of pre- and post-transition baselines. However, simulations of protein unfolding profiles suggest that this method does not work for marginally stable proteins. We show herein that the temperature dependence of the fluorescence intensity of N-acetyl tryptophanamide (NATA) in organic solvents and water may be used to represent the temperature dependence of the fluorescence intensity of tryptophan in native and denatured conformations of a protein, respectively. The wavelength of the emission maximum, lambda (max), of N-acetyl tryptophanamide (NATA) in a particular solvent or tryptophan in proteins is related to the temperature dependence (m) of its fluorescence intensity by the equation: m (K(-1)) = (-0.000299 +/- 2.2 x 10(-5) K(-1) nm(-1)) x lambda (max) (nm) + (0.0919 +/- 0.0025 K(-1)).

Protein fluorescence decay: A gamma function description of thermally induced interconversion of amino acid rotamers

We present a description of fluorescence decay kinetics in complex environments based on gamma functions rather than the conventional approach using exponentials. The gamma function description is tested in measurements on the temperature dependence of the protein human serum albumin (HSA), N-acetyl tryptophanamide (NATA), and 2, 5-dipenyl oxazole (PPO). The monitoring of macromolecular structure and dynamics is demonstrated by means of distinct tryptophan (Trp) rotamer populations and their interconversion in HSA.

Solvation Dynamics of Model Peptides Probed by Terahertz Spectroscopy. Observation of the Onset of Collective Network Motions

We have studied the solvation of model peptides at low hydration levels by terahertz absorption spectroscopy. We have recorded the concentration-dependent terahertz absorption coefficients of N-acetyl-glycine-amide (NAGA), N-acetyl-glycine-methylamide (NAGMA), N-acetyl-leucine-amide (NALA), N-acetyl-leucine-methylamide (NALMA), and N-acetyl-tryptophan-amide (NATA) in aqueous solution. We find a dramatic decrease in the THz absorption, if the number of water molecules per solute is less than 18-20. This change is taken as a signature for the breakdown of peptide-water network motions, which supports the hypothesis that a minimum number of hydration waters is required to activate these motions. This is well below a monolayer coverage of the model peptides. It is interesting to note that the required hydration level corresponds to the number of water molecules which are required for biological functionality.

Characterization of Fluorescent Sterol Binding to Purified Human NPC1

Mutations in the NPC1 gene cause Niemann-Pick type C disease, which appears to result from a defect in intracellular cholesterol trafficking. NPC1 is a member of the resistance-nodulation-cell division (RND) permease superfamily and contains a sterol-sensing domain, yet its cellular function and the identity of its substrates remain unknown. FLAG-tagged human NPC1 was purified from NPC1-expressing Chinese hamster ovary cells by solubilization in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), followed by affinity chromatography. Purified NPC1 in detergent solution appeared to be oligomeric as determined by gel filtration fast protein liquid chromatography and was photolabeled by an azido-cholesterol derivative. Fluorescent cholesterol analogs, including dehydroergosterol, cholestatrienol, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), displayed enhanced fluorescence upon binding to NPC1 and also resulted in saturable, concentration-dependent quenching of NPC1 intrinsic Trp fluorescence. The apparent binding affinity for these three sterols was in the 0.5-6 microm range. Binding of NBD-cholesterol to NPC1 at low detergent concentration (2 mm CHAPS) was of high apparent affinity (0.5-0.6 microm) and occurred rapidly (<1 min). However, binding of a BODIPY-labeled cholesterol derivative was very slow, requiring approximately 3 h to reach equilibrium. The apparent NBD-cholesterol binding affinity was greatly reduced at higher detergent concentration. The stoichiometry of NBD-cholesterol binding to NPC1 was approximately 1. Various sterols, including native cholesterol and 25-hydroxycholesterol, inhibited NBD-cholesterol binding, suggesting that they compete for binding to the protein. Dynamic quenching studies showed that bound NBD-cholesterol was almost completely shielded from the aqueous medium, suggesting that it is buried in a deep hydrophobic pocket in NPC1. The use of fluorescent cholesterol analogs provides novel information on the molecular properties of the sterol-binding site in the full-length NPC1 protein.

Solvochromic Effects in Model Eumelanin Compounds†

We have created an indolic compound which is ideally suited to the study of the relationship between structure and function in eumelanin formation. N-methyl-5-hydroxy-6-methoxyindole (MHMI) is stable in solid and liquid states, highly soluble in a variety of solvents and forms a dimer only through the 4-4' positions. The limited binding possibilities are due to functional groups strategically placed to inhibit chemical interactions through the 2 and 7 positions. It forms a crystal structure with a remarkable packing arrangement, with four monomers grouped in parallel pairs spaced 3.5 A apart within each unit cell. Optical spectra reveal a multi-peaked absorbance profile similar to 5,6-dihydroxyindole (DHI) and N-acetyl-tryptophanamide (NATA), and strong fluorescence emission with radiative quantum yields of 29% and 33% in benzene and acetonitrile, respectively. The quantum yield is similar to that of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and shows that solvent aromaticity by itself does not affect the yield. Solution in chloroform results in an almost complete quenching of the fluorescence but an increase in emission is observed with photoactivation. Crystallographic results shown here suggest new structural possibilities for eumelanin and the controlled binding possibilities make this an excellent model for monitoring changes in function with increasing oligomer size in eumelanin formation.

Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.