Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.

We describe a laboratory exercise that is designed to give advanced undergraduate students in analytical chemistry a meaningful introduction to biological mass spectrometry. We ask the students to solve a mystery: given two proteins, what are they, and from which animals do they originate? This exercise involves use of two mass spectrometers available in our institution: a fairly basic matrix-assisted laser desorption ionization time-of-flight (MALDI–TOF) unit and an electrospray ionization (ESI) ion-trap unit. Our exercise can be completed in two days (including analysis) and provides several interesting and educational surprises. The students, in small groups, are introduced to total mass measurement with ESI, proceed to peptide mass mapping with MALDI–TOF, and finally explore sequence information with ion-trap tandem (fragmentation) mass spectrometry. Even in the most advanced phase, we guide the students, but by this time they usually have their own ideas as to what avenues to pursue. We find that our students become excited after they solve the "mystery"of these proteins, and they receive enough education in the process to be able to join a research project involving biological mass spectrometry. (Less)

Microchip immobilized enzyme reactors (microIMERs) with immobilized endoglucanases were applied for the hydrolysis of methyl cellulose (MC). MCs of various molecular weights were hydrolyzed using two microIMERs containing immobilized celloendoglucanase Cel 5A from Bacillus agaradhaerens (BaCel 5A) connected in series. Hydrolysis by the microIMER could be confirmed from the average molar masses and molar mass distributions measured by size exclusion chromatography (SEC) with online multiangle light scattering and refractive index detection. Methylated cellooligosaccharides with degrees of polymerization (DP) between 1 and 6 formed during hydrolysis were analyzed by direct infusion electrospray ionization ion-trap mass spectrometry (ESI-ITMS). Mass spectra of microIMER- and batch-hydrolyzed samples were compared and no significant differences were found, indicating that microIMER hydrolysis was as efficient as conventional batch hydrolysis. A fast and automated hydrolysis with online MS detection was achieved by connecting the microIMER to high-performance liquid chromatography and ESI-ITMS. This online separation reduced the relative intensities of interfering signals and increased the signal-to-noise ratios in MS. The microIMER hydrolysates were also subjected to SEC interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. With this technique, oligomers with DP 3-30 could be detected. The hydrolysis by the microIMER was performed within 60 min, i.e. significantly faster compared with batch hydrolysis usually performed for at least 24 h. The microIMER also allowed hydrolysis after 10 days of continuous use. The method presented in this work offers new approaches for the analysis of derivatized cellulose and provides the possibility of convenient online, fast, and more versatile analysis compared with the traditional batch method.

Recently, the structure of protein ions in vacuo has received a lot of interest. Experimentally, such structures can only be characterized by global, low-resolution descriptors like the orientationally-averaged collision cross section (sigma) over bar. An alternative descriptor of chain entanglement, the mean overcrossing number (N) over bar, is a very useful tool because it can distinguish sensitively between different conformations based on aspects of their geometry and topology. An overcrossing number distribution arises from an ensemble of different projections of the protein structure onto a viewing plane, where for each projection, the number of crossings by the set of line segments joining sequence-adjacent Calpha atoms is computed. (N) over bar is the mean of this distribution. Despite their usefulness, overcrossing numbers depend on the atomic coordinates and thus cannot be measured for protein ions in vacuo as can collision cross sections. In this work, we explore theoretically whether collision cross sections are correlated with overcrossing numbers. Using a group of diverse proteins with the same chain length as lysozyme and known X-ray structures, we find a weak correlation between (sigma) over bar and (N) over bar. Thus, a nontrivial range of orientationally-averaged cross section values can be consistent with the same mean overcrossing number. Since (N) over bar comprises a very sensitive tool for the study of conformational rearrangements, we checked also for correlations between (sigma) over bar and (N) over bar during molecular dynamics simulated unfolding and relaxation pathways of lysozyme in vacuo. For a given pathway, the two quantities are found to be highly correlated. Although neither (sigma) over bar nor (N) over bar maps one-to-one onto a particular tertiary fold, they are both sensitive descriptors to monitor the conformational state of a protein along a pathway of dynamical change.

The unfolding of protein ions in gas phase (or a vacuum) proceeds rapidly once the total charge reaches a critical value q∗. The actual q∗ value depends on temperature, protein composition, and force field parameters. Here, using molecular dynamics simulations, we explore the onset of the unfolding transition at q>q∗ and its dependence on the nonbonded interaction between monomers. Using lysozyme, we show that, although a loss of attractive cohesion reduces the critical charge for unfolding, the mechanism for the transition may remain essentially unchanged over a range of Coulombic and van der Waals interactions.

Chain entanglement and compactness are two a priori independent properties that convey large-scale shape features of polymer conformations. In this work, we use these properties to monitor the initial step for the in vacuo unfolding of charged lysozyme ions. Using molecular dynamics simulations and a statistical model for the protein charge distribution, we show the existence of a narrow range of total charge within which compact (quasinative) and extended (partly unfolded) conformers can coexist. Denatured conformers are always found above the critical charge for unfolding, whereas quasinative structures are associated with low-charge states. We find that the global molecular shape of the accessible conformers is conserved over a range of temperatures, despite a shift in the critical charge as temperature increases. Within our model, in vacuo unfolding occurs for lysozyme ions with charges +7 and +8, when a thermal bath of T = 500 K is considered. This result is compatible with gas-phase experiments on lysozyme and suggests that unfolding results from the combined effect of heating and the Coulomb repulsion between charged residues.

With the aim of understanding solvent effects in protein folding, unfolding, stability and dynamic behavior, studies of protein ions in vacuo have become popular in recent years. One experimental descriptor which gives a general overview of ionic structure is the orientationally-averaged collision cross section sigma(avg), which is obtained from ion drift mobility (IDM) and other kinds of measurements. In modelling protein structures in vacuo with molecular dynamics simulations, it is necessary to calculate sigma(avg) for a plurality of model structures for comparison with experiments, The collision cross section is sensitive to the roughness (concavity) of the protein surface because of the possibility of multiple collisions during an encounter between a given bath gas particle and the protein. Calculations of sigma(avg), though in principle straightforward, are time consuming, In the work presented below, it was investigated whether a more efficient calculation scheme can be employed without sacrificing too much accuracy. In the new scheme, atomic-scale protein surface granularity is smoothed out by a collected-atoms approach, while large-scale concavity of the protein is essentially preserved.

Tracks induced on surfaces of layered (gypsum, mica) and nonlayered (lithium fluoride, fluorite) crystals by Sn ions with energy of 1 MeV/u, Pb ions with energy of 4.46 MeV/u, and C60 ions with energy of 0.03 MeV/u were studied by the method of shadow replica electron microscopy. The irradiations were carried out at angles of 0.5°, 1° or 2° relative to the surface plane of the crystals. Lengths of two kinds of tracks were compared: (1) surface tracks which are formed on a clean crystal surface; and (2) island tracks which are formed in an island film of gold deposited on the crystal surface prior to irradiation. The following points were established: (1) the lengths of both kinds of tracks are greater on the surfaces of layered crystals than on the surfaces of nonlayered crystals; (2) at angle of irradiation of 0.5° formation of a surface track is initiated at a point where the ion has not yet penetrated the surface plane, but rather moves above the surface plane at a height of ⩽1 nm; and (3) tracks induced by C60 are smaller than tracks induced by monatomic ions. The possible track formation mechanisms are discussed.

Mounting evidence from experiments suggests that the native fold in solution is metastable in dehydrated proteins. Results from a number of experiments that use mass spectrometry indicate also that folding-unfolding transitions take place in protein ions even in the absence of water. These observations on anhydrous proteins call for a re-evaluation of our understanding of the folding transition. In this context, computer-assisted simulations are an important complementary tool. Here, we provide an overview of recent progress on the simulation of proteins in vacuo. In particular, we discuss the response of proteins and protein ions to perturbations that trigger unfolding and re-folding transitions. By comparing the general patterns emerging from theory and experiment, we propose a series of new measurements that could help to validate, and improve, current simulation models.

Molecular dynamics (MD) techniques have been employed to address the structural evolution of highly charged disulfide-bond-reduced lysozyme (DR-LYZ) in vacuo, starting from a compact, nativelike conformation. The results obtained are discussed in terms of factors that promote the stability of lysozyme and are contrasted with the structural evolution of disulfide-bond-intact lysozyme (DI-LYZ), simulated under the same conditions (Reimann et al., Phys. Rev. E, 1999, 60, 7277-7284). For DR-LYS, at least three types of unfolded structures were observed: A, expanded but near-native conformations; B, conformations with the C-terminus portion of the traditionally denoted alpha domain (alpha(2)) extended; and C, conformations that: were overall extended. Generally, higher charge states led to enhanced unfolding, but the charge-state threshold for achieving a certain degree of unfolding depended on temperature and charging configuration. Similar patterns of charge self-solvation were observed in many of the unfolded structures. The beta domain, along with the N-terminus portion of the alpha domain (alpha(1)), emerge as robust structural features which were stabilized both by their own internal hydrogen and self-solvation bonds, as well as by their interactions with each other and with portions of alpha(2).

Starting from a partly unfolded conformer of in vacuo lysozyme, we study the configurational transitions and molecular shape changes that accompany the relaxation (and eventual refolding) of the protein. In particular, we explore the effect of a variable monomer-monomer interaction on the folding dynamics within an ensemble of relaxation trajectories. We find that a strong attractive potential does not necessarily produce configurational "freezing," but instead can be consistent with nativelike refolding. In contrast, a reduction in attraction below a critical value suppresses polymer collapse and eventually leads to complete unfolding. Our results suggest that, qualitatively, folding behavior may not be strongly dependent on the details of the model force field, but rather a feature associated with a range of potential energy functions.

Etch pit edges or walls, induced on the surface of highly-oriented pyrolytic graphite (HOPG) by oxidative etching at high temperature, comprise a potentially useful model for the active surface sites present on various carbon-based materials. We have studied the interactions between the etch-pit edges and nanometer-sized probe tips using various modes of scanning force microscopy (SFM). The etch-pit edges displayed a similar to 100% increase of the friction force, a similar to 20% increase of the adhesion force of the probe tip, and thus similar to 80% increase of the friction coefficient, compared to unmodified basal plane. In addition to the friction force, a topography-induced lateral force is present at etch-pit edges. This force shows a cosine dependence on the angle between the tip-scanning direction and the normal of the etch-pit side wall curvature, when the tip steps upward from the etch pit to the basal plane. In the non-contact mode (small cantilever oscillation amplitude) evidence for enhanced attraction at etch-pit edges was found that could not be observed in tapping mode (large cantilever oscillation amplitude). Our results show that different modes of SFM provide complementary information on surface topography and variations in surface chemistry on the nanometer scale.

The availability of experimental data on biomolecular ions diffusing in a low-pressure gas has raised a number of important questions about the folding behaviour of anhydrous proteins in acuo. In this work, we explore an important aspect of the folding mechanism for anhydrous proteins, namely, its sensitivity to changes in primary sequence. To this end, we study the computer-simulated relaxation dynamics of protein conformers that share the same initial unfolded backbone geometry, but that differ in the primary sequences. The initial unfolded (transient) conformers are derived from an in acuo unfolding run of lysozyme. The relaxation behaviour of unfolded disulfide-intact lysozyme is compared with that of four other different sequences threaded to the same unfolded backbone geometry: disulfide-reduced lysozyme, cytochrome c′, polyglycine and polyalanine. Using a large ensemble of molecular dynamics trajectories, we monitor configurational transitions in a two-dimensional space of order parameters that convey changes in compactness and chain entanglement. Our results indicate that both disulfide-intact and disulfide-reduced lysozyme relax to structures with quasi-native compactness and entanglement. However, fast refolding appears to be more efficient in the presence of the disulfide bridges, since noncompact intermediates persist longer in disulfide-reduced lysozyme. The cytochrome c′ sequence threaded onto the lysozyme transient shows similar relaxation behaviour to that of disulfide-intact lysozyme. Yet, the cytochrome c′ sequence gives rise to several long-lived intermediates, one of which displays global molecular shape features similar to those of native cytochrome c′. In contrast, the relaxation of the polyglycine transient exhibits no initial large-scale collapse, but rather resembles the “pearling” transition of homopolymers (i.e., the initial formation of small locally compact blobs of chain). Polyalanine displays an intermediate behaviour, characterized by instances of both successful and frustrated global collapse. These findings shed light on how primary sequence affects specifically the formation of initial, persistent folding intermediates in acuo.

We present a sensitive analysis technique to explore the configurational space of non-stationary systems. With it, we discuss the pattern of large-scale molecular shape transitions observed during the computer-simulated unfolding of charged in vacuo lysozyme. Using a statistical treatment for the charge distribution among residues, we have estimated the value of the critical total charge that unleashes rapid unfolding in vacuo. Analyses of the molecular dynamics trajectories in a space of molecular shape descriptors indicate a transition between quasi-native folds and denatured conformers. At the critical charge, we find an intermediate regime of persistent partly unfolded structures. This behaviour is compatible with experimental observations of lysozyme ions in the gas phase. Our results indicate that there is a subtle balance between the unfolding tendency of the Coulomb repulsion and the tendency to implode associated with the in vacuo boundary conditions.

Tracks induced on a muscovite mica surface by Sn ions with energy of 1 MeV u−1, and C60 ions with energy of 30 keV u−1, were studied by methods of electron microscopy and scanning force microscopy. The irradiations were carried out at an angle of 2° relative to the surface of the mica. It is shown that Sn ion tracks and C60 ion tracks differ both in shape and size. It is hypothesized that the differences are connected not only with the different density of energy deposited by these ions, but also with disintegration of C60 ions into separate clusters after impact.

We show that the relaxation dynamics of unfolded in vacuo lysozyme is not random. Analyses of molecular dynamics trajectories in a convenient space of molecular shape descriptors reveal a ``favored'' pattern of transitions leading to stable conformations. The relaxation paths exhibit a balanced change in shape features: globular spheroids are formed slowly enough to allow the proper entanglement of secondary-structural elements. The present study shows that a protein in vacuo can actually (re)fold into native and quasinative structures. The driving force for these transformations is intrinsic to the polypeptide chain.

Molecular dynamics simulations were used to interpret a variety of experimental data on highly charged disulfide-bond-intact lysozyme in vacuo. The simulation approach involved submitting a model of the protein [Reimann, Velázquez, and Tapia, J. Phys. Chem. B 102, 9344 (1998)] in a given charge state to a 3-ns-long heat pulse (usually at 500 K) followed by cooling or relaxation for 1 ns back to room temperature (293 K). This treatment yielded a charge threshold around Q(0)=8+ for obtaining significant unfolding, as indicated by an enhancement in collision cross section and conformer length. The collision cross sections and lengths theoretically obtained, along with the threshold charge state for initiating unfolding, were compatible with experimental results on lysozyme in vacuo. The unfolded, highly elongated conformations obtained for Q> or = 9+ displayed a significant level of non-native beta-sheet content which appeared to be additionally stabilized by charge self-solvation.

Unfolding and refolding processes for proteins in vacuo and gas phase are becoming the subject of experimental and theoretical attention. Recently, a molecular dynamics study of unfolding of disulfide-bond-intact lysozyme (DI-LYZ) in vacuo showed large-scale conformational changes (Reimann et al., J. Phys. Chem. B 1998, 102, 2277), thereby providing a configurational space for a denatured state. Here we study the opposite process: from the multimodal unfolding trajectory, a variety of conformations were selected for relaxation studies aimed at computationally mimicking "renaturing" conditions. For DI-LYZ, the relaxations gathered in two distinct classes of conformers as measured by their root-mean-square deviations (RMSD) from the X-ray structure. Structures originating from above the approximate midpoint of the main unfolding transition, with initial RMSD ranging from 8 to 17 Angstrom, relaxed toward persistent compact structures having RMSD approximate to 7.5 +/- 0.5 Angstrom (Class I). They represent compact denatured albeit folded structures. Structures originating from conformers having initial RMSD < 8 Angstrom yielded two subclasses of structures on relaxation: near-native (RMSD approximate to 3 Angstrom) and "nativelike" (RMSD < 2 Angstrom) (together comprising Class II). Both compact and elongated lysozyme species, reported in this work, are consistent with experimentally observed lysozyme conformers in vacuo. The relaxations under renaturation conditions do not elicit a random search of the conformational space. Rather, compact conformers with persistent tertiary and rich secondary structures rapidly form. Therefore, results of the work reported here and in a companion paper (Arteca et al., Phys. Rev. E 1999, 59, 5981) suggest that lysozyme undergoes a folding process in vacuo.