Picosecond time-resolved X-ray crystallography: probing protein function in real time

A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysis-induced structural evolution of wild-type and L29F myoglobin over times ranging from 100 ps to 3 micros. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally "watch" the protein as it executes its function.     

The influence of X-rays on the structural studies of peroxide-derived myoglobin intermediates

In recent years, the awareness of potential radiation damage of metal centers in protein crystals during crystallographic data collection has received increasing attention. The radiation damage can lead to radiation-induced changes and reduction of the metal sites. One of the research fields where these concerns have been comprehensively addressed is the study of the reaction intermediates of the heme peroxidase and oxygenase reaction cycles. For both the resting states and the high-valent intermediates, the X-rays used in the structure determination have given undesired side effects through radiation-induced changes to the trapped intermediates. However, X-rays have been used to generate and trap the peroxy/hydroperoxy state in crystals. In this review, the structural work and the influence of X-rays on these intermediates in myoglobin are summarized and viewed in light of analogous studies on similar intermediates in peroxidases and oxygenases.     

From metmyoglobin to deoxy myoglobin: relaxations of an intermediate state

Metmyoglobin has been reduced at low temperature (below 100 K) using x-rays or by excitation of tris(2,2′,bipyridine)ruthenium(II) chloride with visible light. Upon reduction, an intermediate state is formed where the structure of the protein is very similar to that of metmyoglobin with the water molecule still bound to the heme iron, but the iron is II low spin. The nature of the intermediate state has been investigated with optical spectroscopy. The Q_o and Q_v bands of the intermediate state are split, suggesting that the protoporphyrin is distorted. The intermediate state undergoes a relaxation observed by a shifting of the Soret band at temperatures above 80 K. Above 140 K, the protein begins to relax to the deoxy conformation. The relaxation kinetics of the protein have been monitored optically as a function of time and temperature from minutes to several hours and from 150 K to 190 K. By measuring the entire visible spectrum, we are able to distinguish between electron transfer processes and the protein relaxation from the intermediate state to deoxy myoglobin. The relaxation has been measured in both horse myoglobin and sperm whale myoglobin with the relaxation occurring on faster time scales in horse myoglobin. Both the reduction kinetics and the relaxation show non-exponential behavior. The reduction kinetics can be fit well to a stretched exponential. The structural relaxation from the intermediate state to the deoxy conformation shows a more complex, dynamical behavior and the reaction is most likely affected by the relaxation of the protein within the intermediate state. Received: 30 June 1997 / Accepted: 6 November 1997     

Low temperature X-ray investigation of structural distributions in myoglobin

The results of X-ray structure analysis of metmyoglobin at 300 K, 185 K, 165 K, 115 K and 80 K are reported. The lattice vectorsa andb decrease linearly with temperature whilec shows non-linearity above 180 K, indicating some type of phase transition. Cooling does change the myoglobin structure but only within the structural distribution as determined by individual x ² at room temperature. Two residues showed significant alternative positions for sidechains at higher temperatures while only one position is occupied at low temperatures. In the case of LEU 61 a jump between different positions of the side-chain reduces the potential barrier for the entrance of the O₂ molecule to the heme pocket.The mean square displacements, x ², of the individual residues decrease linearly with temperature in most cases, indicating a parabolic envelope for the potential responsible for motions. A separation of rotational and translational disorder of the entire molecule is discussed. Comparison with Mössbauer spectroscopy indicates that protein dynamics on a time scale faster than 10^-7 s is not simply a harmonic process. Extrapolation of the structural distributions toT=0 K shows that a large zero point distribution of the myoglobin structure exists, thus proving that there is no absolute energy minimum for one well defined conformation.Dedicated to Prof. H. Frauenfelder on his 65^th birthday     

Synthesis and DNA-recognition and -cleavage properties of multiply charged porphyrin esters

A series of systematically modified porphyrin esters, compounds 1-6, with multiple, permanent positive charges introduced at the meso-positions via N-methylated 4-, 3-, or 2-pyridyl moieties, were prepared and characterized. Their singlet-oxygen production, CT-DNA-binding properties, and plasmid-DNA photocleavage propensities were determined spectroscopically and by gel electrophoresis, and compared to those of the known, fourfold-charged parent porphyrin 4,4',4',4'-porphyrin-5,10,15,20-tetrayltetrakis(1-methylpyridinium) (TMPyP4). Some interesting structure-activity relationships could be established to rationalize effects affecting DNA binding mode and cleavage ability.     

Evolution of the amino acid substitution in the mammalian myoglobin gene

Summary Multivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.     

Structural stability of myoglobin and glycomyoglobin: a comparative molecular dynamics simulation study

Glycoproteins are formed as the result of enzymatic glycosylation or chemical glycation in the body, and produced in vitro in industrial processes. The covalently attached carbohydrate molecule(s) confer new properties to the protein, including modified stability. In the present study, the structural stability of a glycoprotein form of myoglobin, bearing a glucose unit in the N-terminus, has been compared with its native form by the use of molecular dynamics simulation. Both structures were subjected to temperatures of 300 and 500 K in an aqueous environment for 10 ns. Changes in secondary structures and RMSD were then assessed. An overall higher stability was detected for glycomyoglobin, for which the most stable segments/residues were highlighted and compared with the native form. The simple addition of a covalently bound glucose is suggested to exert its stabilizing effect via increased contacts with surrounding water molecules, as well as a different pattern of interactions with neighbor residues.

Electronic supplementary material

The online version of this article (doi:10.1007/s10867-015-9383-2) contains supplementary material, which is available to authorized users.     

Solvent modulation of the structural heterogeneity in FeIII myoglobin samples: a low temperature EPR investigation

High spin FeIII myoglobin samples in solutions with different solvent composition have been investigated at low temperature by Electron Paramagnetic Resonance spectroscopy. The g = 6 line of the spectrum has been analyzed in terms of a distribution of the two crystal field parameters ₁ and ₂. By means of the Angular Overlap Method, it has been shown that these distributions entail, in turn, a distribution in the iron-heme displacement along the normal to the heme-plane. The spread in this iron-heme distance, which can be connected with the binding action of the proximal histidine, has been proposed as a quantitative measurement of the structural heterogeneity (conformational substate landscape) displayed by the protein molecules. The results point out, moreover, that the solvent composition can affect the structural heterogeneity of the protein system. In particular, addition of glycerol, ethylene glycol and sucrose yields a significant reduction in the spread of the ironheme displacement, while the presence of ammonium sulfate induces a change in the average position of the iron in the heme-plane. The role played by the solvent in the structure and dynamics of the protein, in connection also with the conformational substate distribution, is discussed. Correspondence to: S. Cannistraro     

Effects of carbohydrate headgroups on the stability of induced cubic phases in binary mixtures of glycolipids

This paper is part in a series of papers, investigating the influence of carbohydrate headgroups on the mesogenic properties of glycolipids. While previous papers focussed on the synthesis and mesogenic properties of the pure compounds, we will discuss here our results obtained with binary mixtures. Mixtures of compounds, one forming a lamellar phase and the other one a columnar phase in their pure state, displayed always an induced cubic phase. The stability of this induced cubic phase depends significantly on the structure of the carbohydrate headgroup of both components. Thus it was possible to derive structure–property relationships by comparison of the phase diagrams that have been obtained, if the carbohydrate headgroup of one component was changed systematically. We observed an interesting effect of galactose headgroups which might be of great biological importance. Furthermore, the observed kind of kinetic of the S_A→cub transition might also be of great biological relevance.     

The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe-CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (DeltaV1 = -2.5+/-0.6 ml mol(-1) and DeltaH1 = 8.1+/-3.0 kcal mol(-1)). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster (tau=330 ns) relative to native protein (tau=700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (DeltaV2 = 5.4+/-2.5 ml mol(-1) and DeltaH2 = 0.7+/-2.2 kcal mol(-1)) than those for native Mb (DeltaV2 = 14.3+/-0.8 ml mol(-1) and DeltaH2 = 7.8+/-3.5 kcal mol(-1)). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.     

Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer--formed using a micro-contact imprinting method

Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities.

The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers.

With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03 ± 0.89 × 10⁻¹¹ mole/cm² of template from a 5.68 × 10⁻⁷ M myoglobin solution, compared to 2.58 ± 0.02 × 10⁻¹¹ mole/cm² for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2 wt.% SDS and 0.6 wt.% NaOH used at 80 °C for 30 min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal.

The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (K_d) for the specific binding phase to be 3.4 × 10⁻⁷ M and the binding site capacity to be 7.24 × 10⁻¹¹ mole/cm². For the non-specific binding phase, K_d was found to be 1.355 × 10⁻⁵ M and the binding site capacity was determined as 9.62 × 10⁻¹⁰ mole/cm².

Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68 × 10⁻⁷ M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.     

Challenges at the frontiers of structural biology

Knowledge of the three-dimensional structures of proteins is the key to unlocking the full potential of genomic information. There are two distinct directions along which cutting-edge research in structural biology is currently moving towards this goal. On the one hand, tightly focused long-term research in individual laboratories is leading to the determination of the structures of macromolecular assemblies of ever-increasing size and complexity. On the other hand, large consortia of structural biologists, inspired by the pace of genome sequencing, are developing strategies to determine new protein structures rapidly, so that it will soon be possible to predict reasonably accurate structures for most protein domains. We anticipate that a small number of complex systems, studied in depth, will provide insights across the field of biology with the aid of genome-based comparative structural analysis.     

Secondary metabolites of plants from the genus Saussurea: chemistry and biological activity

The Asteraceae family comprises ca. 1000 genera, mainly distributed in Asia and Europe. Saussurea DC., as the largest subgenus of this family, comprises ca. 400 species worldwide, of which ca. 300 species occur in China. Most plants in China grow wild in the alpine zone of the Qingzang Plateau and adjacent regions at elevations of 4000 m. Plants of the genus Saussurea (Asteraceae) are used in both traditional Chinese folk medicine and Tibet folklore medicine, since they are efficacious in relieving internal heat or fever, harmonizing menstruation, invigorating blood circulation, stopping bleeding, alleviating pain, increasing energy, and curing rheumatic arthritis. A large number of biologically active compounds have been isolated from this genus. This review shows the chemotaxonomy of these compounds (215 compounds) such as sesquiterpenoids (101 compounds), flavonoids (19 compounds), phytosterols (15 compounds), triterpenoids (25 compounds), lignans (32 compounds), phenolics (23 compounds), and chlorophylls (11 compounds). Biological activities (anti‐inflammatory, anticancer, antitumor, hepatoprotective, anti‐ulcer, cholagogic, immunosuppressive, spasmolytic, antimicrobial, antiparasitic, antifeedant, CNS depressant, antioxidant, etc.) of these compounds, including structure–activity relationships, are also discussed.     

Challenges at the frontiers of structural biology

Knowledge of the three-dimensional structures of proteins is the key to unlocking the full potential of genomic information. There are two distinct directions along which cutting-edge research in structural biology is currently moving towards this goal. On the one hand, tightly focused long-term research in individual laboratories is leading to the determination of the structures of macromolecular assemblies of ever-increasing size and complexity. On the other hand, large consortia of structural biologists, inspired by the pace of genome sequencing, are developing strategies to determine new protein structures rapidly, so that it will soon be possible to predict reasonably accurate structures for most protein domains. We anticipate that a small number of complex systems, studied in depth, will provide insights across the field of biology with the aid of genome-based comparative structural analysis.     

Challenges at the frontiers of structural biology

Knowledge of the three-dimensional structures of proteins is the key to unlocking the full potential of genomic information. There are two distinct directions along which cutting-edge research in structural biology is currently moving towards this goal. On the one hand, tightly focused long-term research in individual laboratories is leading to the determination of the structures of macromolecular assemblies of ever-increasing size and complexity. On the other hand, large consortia of structural biologists, inspired by the pace of genome sequencing, are developing strategies to determine new protein structures rapidly, so that it will soon be possible to predict reasonably accurate structures for most protein domains. We anticipate that a small number of complex systems, studied in depth, will provide insights across the field of biology with the aid of genome-based comparative structural analysis.     

Synthesis and structure-activity relationships of 16-modified analogs of 2-methoxyestradiol

A series of 16-modified 2-methoxyestradiol analogs were synthesized and evaluated for antiproliferative activity toward HUVEC and MDA-MB-231 cells, and for susceptibility to conjugation. In addition, the estrogenicity of these analogs was accessed by measuring cell proliferation of the estrogen-dependent cell line MCF7 in response to compound treatment. It was observed that antiproliferative activity dropped as the size of the 16 substituent increased. Selected analogs tested in glucuronidation assays had similar rates of clearance to 2-methoxyestradiol, but had enhanced clearance in sulfonate conjugation assays.     

Active core structure of terfestatin A, a new specific inhibitor of auxin signaling

The auxins, plant hormones, regulate many aspects of the growth and development of plants. Terfestatin A (TrfA), a novel auxin signaling inhibitor, was identified in a screen of Streptomyces sp. F40 extracts for inhibition of the expression of an auxin-inducible gene. However, the mode of action of TrfA has not been elucidated. To identify the active core structure, 25 derivatives of TrfA were synthesized and their inhibitory activities against auxin-inducible gene expression were evaluated. The structure–activity relationships revealed the essential active core structure of TrfA, 3-butoxy-4-methylbiphenyl-2,6-diol, which will lead to the design of biotin-tagged active TrfA.     

Engineered chimeras reveal the structural basis of hexacoordination in globins: A case study of neuroglobin and myoglobin

Background

Myoglobin (Mb) and neuroglobin (Ngb) are representative members of pentacoordinated and bis-histidyl, hexacoordinated globins. In spite of their low sequence identity, they show surprisingly similar three-dimensional folds. The ability of Ngb to form a hexacoordinated bis-histidyl complex with the distal HisE7 has a strong impact on ligand affinity. The factors governing such different behaviors have not been completely understood yet, even though they are extremely relevant to establish structure–function relationships within the globin superfamily.

Methods

In this work we generated chimeric proteins by swapping a previously identified regulatory segment – the CD region – and evaluated comparatively the structural and functional properties of the resulting proteins by molecular dynamics simulations, and spectroscopic and kinetic investigations.

Results

Our results show that chimeric proteins display heme coordination properties displaced towards those expected for the corresponding CD region. In particular, in the absence of exogenous ligands, chimeric Mb is found as a partially hexacoordinated bis-histidyl species, whereas chimeric Ngb shows a lower equilibrium constant for forming a hexacoordinated bis-histidyl species.

Conclusions

While these results confirm the regulatory role of the CD region for bis-histidyl hexacoordination, they also suggest that additional sources contribute to fine tune the equilibrium.General significanceGlobins constitute a ubiquitous group of heme proteins widely found in all kingdoms of life. These findings raise challenging questions regarding the structure–function relationships in these proteins, as bis-histidyl hexacoordination emerges as a novel regulatory mechanism of the physiological function of globins.     

Forging the link between structure and function of electrogenic cotransporters: the renal type IIa Na+/Pi cotransporter as a case study

Electrogenic cotransporters are membrane proteins that use the electrochemical gradient across the cell membrane of a cosubstrate ion, for example Na⁺ or H⁺, to mediate uphill cotransport of a substrate specific to the transport protein. The cotransport process involves recognition of both cosubstrate and substrate and translocation of each species according to a defined stoichiometry. Electrogenicity implies net movement of charges across the membrane in response to the transmembrane voltage and therefore, in addition to isotope flux assays, the cotransport kinetics can be studied in real-time using electrophysiological methods. As well as the cotransport mode, many cotransporters also display a uniport or slippage mode, whereby the cosubstrate ions translocate in the absence of substrate. The current challenge is to define structure–function relationships by identifying functionally important elements in the protein that confer the transport properties and thus contribute to the ultimate goal of having a 3-D model of the protein that conveys both structural and functional information. In this review we focus on a functional approach to meet this challenge, based on a combination of real-time electrophysiological assays, together with molecular biological and biochemical methods. This is illustrated, by way of example, using data obtained by heterologous expression of the renal Na⁺-coupled inorganic phosphate cotransporter (NaP_i-IIa) for which structure–function relationships are beginning to emerge.