Reversible photoregulation of binding of alpha-chymotrypsin to a gold surface

An ability to optically modulate the interactions of surfaces with functional biomolecules provides an important basis for generating new technologies including reversible biosensors, advanced medical implants, and biomolecular computers. Here we report the first example of reversible photoregulation of binding of a protease to a functional surface. A modular approach is presented with a surface-bound inhibitor containing a photoisomerizable azobenzene core to which is attached (i) appropriate protease binding functionality and (ii) a tether for surface attachment. The principle is demonstrated for alpha-chymotrypsin using a phenylalanine-based trifluoromethylketone inhibitor containing an azobenzene core and an alkyne-functionalized ethylene glycol tether, which is attached to the surface using click chemistry. UV/vis irradiation of the functional surface leads to a significant, reversible change in the amount of alpha-chymotrypsin that attaches to the surface, as measured by surface plasmon resonance.     

Cation exchange in lipophilic G-quadruplexes: not all ion binding sites are equal

Lipophilic guanosine derivatives that form G-quadruplexes are promising building blocks for ionophores and ion channels. Herein, cation exchange between solvated cations (K+ and NH4+) and bound cations in the G-quadruplex [G1]16.4Na+.4DNP- was studied by electrospray ionization mass spectrometry and solution 1H, 15N NMR spectroscopy. The ESI-MS and 1H NMR data provided evidence for the formation of mixed-cationic Na+, K+ G-quadruplexes. The use of 15NH4+ cations in NMR titrations, along with 15N-filtered 1H NMR and selective NOE experiments, identified two mixed-cationic intermediates in the cation exchange pathway from [G1]16.4Na+.4DNP- to [G1]16.4NH4+.4DNP-. The central Na+, bound between the two symmetry-related G8-Na+ octamers, exchanges with either K+ or NH4+ before the two outer Na+ ions situated within the C4 symmetric G8 octamers. A structural rationale, based on differences in the cations' octahedral coordination geometries, is proposed to explain the differences in site exchange for these lipophilic G-quadruplexes. Large cations such as Cs+ can be exchanged into the central cation binding site that holds the two symmetry-related C4 symmetric G8 octamer units together. The potential relevance of these findings to both supramolecular chemistry and DNA G-quadruplex structure are discussed.     

Effect of ionic strength on the binding of alpha-chymotrypsin to nanoparticle receptors

Negatively charged carboxylate-functionalized mixed monolayer protected clusters (MMPCs) effectively bind and inhibit alpha-chymotrypsin based on complementary electrostatic surface recognition. We demonstrate that this binding can be disrupted by varying the ionic strength of the medium. Enzyme activity in the presence of MMPCs increases from 5% to 97% of native activity as salt concentration is increased from 0 to 1.5 M. Variation of ionic strengths after complete binding over 13 h results only in a modest restoration of enzymatic activity (< 35%). The conformation of chymotrypsin was characterized using circular dichroism and fluorescence spectroscopy, correlating structure with enzymatic activity. This work provides a useful insight of the electrostatic influence on protein--MMPC interactions and can be applied toward MMPC-based controlled release of proteins in vivo.     

Substrate monolayers as electrochemical sensing elements for alpha-chymotrypsin

A disulfide, which carried two L-phenylalanyl p-nitroanilide (Phe-pNA) moieties at both ends, was prepared by the coupling of 11,11'-dithiodiundecanoic acid (DTUA) with Phe-pNA. The compound obtained (DTUA-Phe-pNA) formed a self-assembled monolayer (SAM) on a gold electrode and vacuum-evaporated gold thin film as proven by cyclic voltammetry, and reflection absorption spectroscopy, respectively. Incubation of alpha-chymotrypsin with the SAM-modified electrode induced both a decrease in anodic and cathodic peak currents (-DeltaIa and -DeltaIc) and an increase in potential difference (DeltaEp) in the cyclic voltammogram of potassium ferricyanide as a probe, which suggested the attack of the enzyme at the amide group between Phe and pNA groups of the SAM, resulting in the formation of an intermediate. The linear relationship between the initial rate of increase in the amount of enzyme bound to the SAM and in both DeltaIa (absolute value of the decrease in anodic peak current) and DeltaEp values was confirmed by the quartz crystal microbalance method. The binding rate of the enzyme to the Phe-pNA SAM was dependent on the surface density of the Phe-pNA group in the SAM. The alpha-chymotrypsin-induced increases in the DeltaIa and DeltaEp values were inhibited by the addition of N-acetyl-D-phenylalanine methyl ester (N-Ac-D-Phe-OMe). In contrast with alpha-chymotrypsin, trypsin did not show a significant increase in the DeltaIa and DeltaEp values upon incubation with Phe-pNA-carrying SAM. These results could be attributed to the specific attack of alpha-chymotrypsin to the amide group in the SAM. The inhibition constant for N-Ac-D-Phe-OMe in the SAM system was quite similar to that in the free substrate system, showing that the enzymatic reaction above the SAM proceeds in a similar way to that in the homogeneous solution system.     

Nitrogen balance and delta15N: why you're not what you eat during pregnancy

Carbon (13C/12C) and nitrogen (15N/14N) stable isotope ratios were longitudinally measured in human hair that reflected the period from pre-conception to delivery in 10 pregnant women. There was no significant change in the delta13C results, but all subjects showed a decrease in delta15N values (-0.3 to -1.1 per thousand) during gestation. The mechanisms causing this decrease in hair delta15N have not been fully elucidated. However, since the delta15N values of dietary nitrogen and urea nitrogen are significantly lower compared to maternal tissues, it is hypothesized that the increased utilization of dietary and urea nitrogen for tissue synthesis during pregnancy resulted in a reduction of the steady state diet to a body trophic level effect by approximately 0.5-1 per thousand. An inverse correlation (R2 = 0.67) between hair delta15N and weight gain was also found, suggesting that positive nitrogen balance results in a reduction of delta15N values independent of diet. These results indicate that delta15N measurements have the ability to monitor not only dietary inputs, but also the nitrogen balance of an organism. A potential application of this technique is the detection of fertility patterns in modern and ancient species that have tissues that linearly record stable isotope ratios through time.     

A statistical approach to delta layer depth profiling

We present a simple statistical model describing the removal and relocation of material during a sputter depth profiling experiment. All input parameters are determined from low‐fluence molecular dynamics simulations, making the model de facto parameter free. The model can be used to extrapolate data from the molecular dynamics simulations to projectile fluences relevant to sputter depth profiling experiments. As a result, the erosion of the surface is calculated in terms of fluence‐dependent filling factors of different sample layers. Using input data determined for the 20‐keV C₆₀ cluster bombardment of silicon, it is found that a steady‐state erosion profile is reached after removal of approximately 20 monolayer equivalents of material. Plotting the contribution of particles from a specific layer to the instantaneous sputtered flux, one can directly determine the delta layer response function predicted from such a model. It is shown that this function can be parameterized by the semiempirical Dowsett response function, and the resulting fitting parameters are compared with published depth profile data. The model is then used to study the role of different processes influencing the observed depth resolution. We find that the statistical nature of the sputtering process suffices to explain many features of experimentally measured delta layer depth profiles. Copyright © 2012 John Wiley & Sons, Ltd.     

Nitrogen balance and delta15N: why you're not what you eat during nutritional stress

While past experiments on animals, birds, fish, and insects have shown changes in stable isotope ratios due to nutritional stress, there has been little research on this topic in humans. To address this issue, a small pilot study was conducted. Hair samples from eight pregnant women who experienced nutritional stress associated with the nausea and vomiting of morning sickness (hyperemesis gravidarum) were measured for carbon (delta13C) and nitrogen (delta15N) stable isotope ratios. The delta13C results showed no change during morning sickness or pregnancy when compared with pre-pregnancy values. In contrast, the delta15N values generally increased during periods of weight loss and/or restricted weight gain associated with morning sickness. With weight gain and recovery from nutritional stress, the hair delta15N values displayed a decreasing trend over the course of gestation towards birth. This study illustrates how delta15N values are not only affected by diet, but also by the nitrogen balance of an individual. Potential applications of this research include the development of diagnostic techniques for tracking eating disorders, disease states, and nitrogen balance in archaeological, medical, and forensic cases.     

Tracking radical migration in large hydrogen deficient peptides with covalent labels: Facile movement does not equal indiscriminate fragmentation

Photodissociation of iodo-tyrosine modified peptides yields localized radicals on the tyrosine side chain, which can be further dissociated by collisional activation. We have performed extensive experiments on model peptides, RGYALG, RGYG, and their derivatives, to elucidate the mechanisms underlying backbone fragmentation at tyrosine. Neither acetylation nor deuteration of the tyrosyl phenolic hydrogen significantly affects backbone fragmentation. However, deuterium migration from the tyrosyl β carbon is concomitant with cleavage at tyrosine. Substitution of tyrosine with 4-hydroxyphenylglycine, which does not have β hydrogens, results in almost complete elimination of backbone fragmentation at tyrosine. These results suggest that a radical situated on the β carbon is required for a-type fragmentation in hydrogen-deficient radical peptides. Replacement of the αH of the residue adjacent to tyrosine with methyl groups results in significant diminution of backbone fragmentation. The initial radical abstracts an αH from the adjacent amino acid, which is poised to “rebound” and abstract the βH of tyrosine through a six-membered transition-state. Subsequent β-scission leads to the observed a-type backbone fragment. These results from deuterated peptides clearly reveal that radical migration in peptides can occur and that multiple migrations are not infrequent. Counterintuitively, close examination of all experimental results reveals that the probability for fragmentation at a particular residue is well correlated with thermodynamic radical stability. A-type fragmentation therefore appears to be most likely when favorable thermodynamics are combined with the relevant kinetic control. These results are consistent with ab initio calculations, which demonstrate that barriers to migration are significantly smaller in magnitude than probable dissociation thresholds.     

Ultrafast surface solvation dynamics and functionality of an enzyme alpha-chymotrypsin upon interfacial binding to a cationic micelle

In this contribution we report studies on enzymatic activity of alpha-chymotrypsin (CHT) upon complexation with cationic cetyltrimethylammonium bromide (CTAB) micelle. With picosecond time resolution, we examined solvation dynamics at the interface of CHT-micelle complex, and rigidity of the binding. We have used 5-(dimethyl amino) naphthalene-1-sulfonyl chloride (dansyl chloride; DC) that is covalently attached to the enzyme at the surface sites. The solvation processes at the surface of CHT in buffer solution are found to be mostly in the sub-50 ps time scale. However, at the interface the solvation correlation function decays with time constant 150 ps (65%) and 500 ps (35%), which is significantly different from those found at the enzyme and micellar surfaces. The binding structure of the enzyme-micelle complex was examined by local orientational motion of the probe DC and compared with the case without micelle. The orientational dynamics of the probe DC in the complex reveals a structural perturbation at the surface sites of CHT upon complexation, consistent with other reported structural studies. We also found possible entanglement of charge transfer dynamics of the probe DC on the measured solvation processes by using time-resolved area normalized emission spectroscopic technique. The interfacial solvation process and complex rigidity elucidate the strong recognition mechanism between CHT and the micelle, which is important to understand the biological function of CHT upon complexation with the micelle.     

Probing residual interactions in unfolded protein states using NMR spin relaxation techniques: an application to delta131delta

Residual interactions in delta131delta, a large disordered fragment of staphylococcal nuclease, have been probed at two different pHs using backbone (15)N and side-chain methyl (2)H NMR spin relaxation techniques. The amplitudes of picosecond time-scale motions of both the backbone and side chains do not change considerably at either pH value, although they are significantly larger than those observed for folded proteins. In contrast, dramatic increases in the amplitudes of motions occurring on a nanosecond time scale are observed throughout delta131delta at pH 3 relative to pH 5. This is consistent with a picture in which residual hydrophobic contacts at pH 5 are disrupted by electrostatic repulsions that dominate at the lower pH.     

Tunable inhibition and denaturation of alpha-chymotrypsin with amino acid-functionalized gold nanoparticles

Water-soluble gold nanoparticles bearing diverse l-amino acid terminals have been fabricated to probe the effect of receptor surface on protein surface binding. The interaction of these nanoparticles with alpha-chymotrypsin (ChT) was investigated by activity assay, gel electrophoresis, zeta-potential, circular dichroism, and fluorescence spectroscopy. The results show that both electrostatic and hydrophobic interactions between the hydrophobic patches of receptors and the protein contribute to the stability of the complex. The microscopic binding constants for these receptor-protein systems are 10(6)-10(7) M(-1), with the capacity of the nanoparticle receptors to bind proteins determined by both their surface area and their surface charge density. Furthermore, it is found that the hydrophilic side chains destabilize the ChT structure through either competitive hydrogen bonding or breakage of salt bridges, whereas denaturation was much slower with hydrophobic amino acid side chains. Significantly, correlation between the hydrophobicity index of amino acid side chains and the binding affinity and denaturation rates was observed.     

Modulation of the catalytic behavior of alpha-chymotrypsin at monolayer-protected nanoparticle surfaces

Amino-acid-functionalized gold clusters modulate the catalytic behavior of alpha-chymotrypsin (ChT) toward cationic, neutral, and anionic substrates. Kinetic studies reveal that the substrate specificity (k(cat)/K(M)) of ChT-nanoparticle complexes increases by approximately 3-fold for the cationic substrate but decreases by 95% for the anionic substrate as compared with that of free ChT, providing enhanced substrate selectivity. Concurrently, the catalytic constants (k(cat)) of ChT show slight augmentation for the cationic substrate and significant attenuation for the anionic substrate in the presence of amino-acid-functionalized nanoparticles. The amino acid monolayer on the nanoparticle is proposed to control both the capture of substrate by the active site and release of product through electrostatic interactions, leading to the observed substrate specificities and catalytic constants.