The energy landscapes and motions of proteins.

Recent experiments, advances in theory, and analogies to other complex systems such as glasses and spin glasses yield insight into protein dynamics. The basis of the understanding is the observation that the energy landscape is complex: Proteins can assume a large number of nearly isoenergetic conformations (conformational substates). The concepts that emerge from studies of the conformational substates and the motions between them permit a quantitative discussion of one simple reaction, the binding of small ligands such as carbon monoxide to myoglobin.

Probing the mechanisms of macromolecular recognition: the cytochrome b5-cytochrome c complex.

The specificity of complex formation between cytochrome b5 (cyt b5) and cytochrome c (cyt c) is believed to involve the formation of salt linkages between specific carboxylic acid residues of cyt b5 with lysine residues on cyt c. Site-directed mutagenesis was used to alter the specified acidic residues of cyt b5 to the corresponding amide analogues, which resulted in a lower affinity for complex formation with cyt c. The dissociation of the complex under high pressure resulted in specific volume changes, the magnitude of which reflected the degree of solvation of the acidic residues in the proposed protein-protein interface.

The catalytic pathway of cytochrome p450cam at atomic resolution.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy.

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths, with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm(-1), which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.

Role of NADPH:cytochrome c reductase and DT-diaphorase in the biotransformation of mitomycin C1.

Hypoxic cells of solid tumors are difficult to eradicate by X-irradiation or chemotherapy; as an approach to this problem, our laboratories are investigating the effects of the bioreductive alkylating agent mitomycin C (MC) on hypoxic cells. This antibiotic was preferentially toxic to EMT6 mouse mammary tumor cells and V79 Chinese hamster lung fibroblasts under hypoxic conditions, but it was equitoxic to Chinese hamster ovary cells in the presence and absence of oxygen. All cell lines catalyzed the formation of reactive metabolites under hypoxic conditions and contained NADPH:cytochrome c reductase and DT-diaphorase, two enzymes which may be responsible for the cellular activation of MC. Although a correlation existed between enzymatic activities and the formation of reactive metabolites from MC, there was no correspondence between these parameters and the degree of cytotoxicity expressed by MC under hypoxic conditions. Purified NADPH:cytochrome c reductase reduced MC in the absence of oxygen, with addition of cytochrome P-450 enhancing, but not participating directly in, the reduction reaction. Addition of NADP+ to cell sonicates substantially reduced NADPH:cytochrome c reductase activity, while the formation of reactive metabolites was affected only slightly; converse results were observed using mersalyl. Exposure of cell sonicates to dicumarol inhibited DT-diaphorase activity, while the rate of formation of reactive metabolites of MC was enhanced. The findings suggest that NADPH:cytochrome c reductase and some as yet to be identified enzyme(s) are important for the reductive activation of MC. DT-diaphorase and cytochrome P-450 are not directly involved in the activation of MC, but they appear to modulate the degree of activation to reactive species, which are presumably responsible for the observed cytotoxicity.

Experimental and theoretical high pressure strategies for investigating protein-nucleic acid assemblies.

A method was developed to investigate the stability of protein-nucleic acid complexes using hydrostatic pressure during electrophoretic gel mobility shift analysis. The initial system probed by this technique was the well-characterized cognate BamHI-DNA complex. Band shift analysis at several elevated pressures found the equilibrium dissociation (K(d)) constant to be dependent on pressure, which allowed the volume change of dissociation (deltaV) to be calculated. In order to describe the effects of pressure on the specific BamHI-DNA complex at the molecular level, molecular dynamics simulations at both ambient and elevated pressure was performed. Comparison of the simulation trajectories identified several individual BamHI-DNA contacts that are disrupted due to pressure. The disruption of these contacts can be attributed to an observed pressure-induced increase in hydration at the protein-DNA interface during the elevated pressure simulation.

Interpretation of hyperfine shift patterns in ferricytochromes b5 in terms of angular position of the heme: a sensitive probe for peripheral heme protein interactions.

The 1H-NMR hyperfine shift pattern of the heme in a variety of low-spin ferricytochromes b5 has been analyzed in terms of the angular position of the prosthetic group within a structurally and magnetically-conserved protein matrix. A simple model is presented in which the changes in the spread of the predominantly contact shifted methyl and predominantly dipolar shifted meso-H signals of the heme, as well as shift trends for individual signals, provide sensitive indicators of the orientation of the heme relative to the orbital hole (singly-occupied d orbital), which in turn is related to the rhombic magnetic axes. The invariance of the axial His and non-coordinated residue hyperfine shifts show that it is the heme within a relatively rigid protein matrix, rather than the magnetic coordinate system, which is displaced angularly about the heme normal in order to accommodate variations in the polypeptide, orientation of the heme about the alpha,gamma-meso axis, and the length of heme carboxylate chains. Native heme shows increased counterclockwise rotation about the heme normal in the order rat-->beef-->chicken ferricytochrome b5, which is attributed largely to increased bulk of a variable sequence hydrophobic cluster consisting of residues 23, 25 and 32. The two alternate heme orientations about the alpha,gamma-meso axis are shown to also differ by rotation about the heme normal. A semiquantitative estimate of the degree of angular accommodation based on the spread of the meso-H rhombic dipolar shifts indicate rotations of 2-10 degrees. Possible functional consequences of such angular accommodation in relation to the role of these proteins in electron transfer are discussed.

Molecular recognition mediated by bound water. A mechanism for star activity of the restriction endonuclease EcoRI.

Many restriction endonucleases such as EcoRI lose some specificity for their recognition sequence under certain buffer conditions. The cause of this disruption of accurate protein-DNA recognition has never been explained. By cleaving DNA with EcoRI in the presence of several osmolytes, we show that the extent of this EcoRI "star activity" depends strongly upon osmotic pressure. The loss of specificity accompanying decreased water activity implies a role for one or more water molecules in recognition of specific sequences of DNA. Water mediation may constitute a general motif for sequence-specific DNA recognition by restriction enzymes and other DNA-binding proteins.

Mapping electrostatic interactions in macromolecular associations.

In the association of electron transfer proteins, electrostatics has been proposed to play a role in maintaining the stability and specificity of the biomolecular complexes formed. An excellent model system is the interaction between mammalian cytochrome b5 and cytochrome c, in which the X-ray structures of the individual components reveal a complementary asymmetry of charges surrounding their respective redox centers. Determining the exact extent of the electrostatic interactions and identifying the specific residues involved in the formation of the electron transfer complex has proved more elusive. We report herein the utilization of high-pressure techniques, together with site-directed mutagenesis, to provide a map of the interaction domains in biomolecular complex formation. The application of high pressure disrupts macromolecular associations since dissociation of the complex results in a decreased volume of the system due to the solvation of charges that had been previously sequestered in the interface region and force solvation of hydrophobic surfaces. Site-directed mutagenesis of a totally synthetic gene for rat liver cytochrome b5, which expresses this mammalian protein in Escherichia coli as a hemecontaining soluble component, was used to selectively alter negatively charged residues of cytochrome b5 to neutral amide side-chains. We have demonstrated that the interaction domain of cytochrome b5 with cytochrome c can be mapped from a comparison of dissociation volumes of these modified cytochrome b5-cytochrome c complexes with the native complex. Using these techniques we can specifically investigate the role of particular residues in the equilibrium association of these two electron transfer proteins. Single-point mutations in the interaction domain give nearly identical effects on the measured dissociation volumes, yet removal of acidic residues outside the recognition surface yield volumes similar to wild-type protein. Multiple mutations in the proposed protein-protein interaction site are found to allow greater solvent-accessibility of the interface as reflected in a diminution in the volume changes on subsequent charge removal. This is indicative that the interprotein salt-bridges in this complex provide a mechanism for a greater exclusion of solvent from the interfacial domain of the complex, resulting in a more stable association.

Nature's universal oxygenases: the cytochromes P450.

Cytochromes P450 are utilized in an enormous diversity of biological reactions, including degradation of xenobiotics, generation of hormones and biosynthesis of a variety of important biological compounds. The cytochrome P450 family is a major participant in nearly all metabolism of pharmaceutical reagents. The presence of different P450 enzymes in various quantities in individuals makes the prediction of drug responses in patients highly complex. A large literature describing mechanistic studies has characterized several intermediates in the oxygenation pathway. It has recently been shown that two or more possible oxygenated forms of the P450 haem can participate in various oxygenations, with some intermediates being highly electrophilic and others being nucleophilic.

Protein engineering for molecular electronics.

Recombinant DNA technology allows the manipulation of the physical properties of proteins that perform electron transport and photochemical processes. Recent work is reviewed that has a potential impact on the development of molecular electronic devices, within a general framework outlining strategies for device fabrication. This review is also published in Current Opinion in Structural Biology 1992, 2:587-592.

Engineering cytochrome P450s for bioremediation.

Building on the vast knowledge of active site structure and catalytic mechanisms of the P450 monooxygenase systems, significant efforts to utilize the rational design of engineered P450s are emerging as an approach to solve the problems of bioremediation. P450 enzymes are being designed to alter substrate specificities and catalytic efficiency in predefined ways. In addition, random mutagenesis and in vitro evolution are being considered as exciting methods for generating mutant P450s with increased bioremediation abilities.

Electrostatic stabilization in four-helix bundle proteins.

Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of alpha-helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.

Participation of water in Hin recombinase--DNA recognition.

The participation of water molecules in the interaction between the Hin recombinase and its operator DNA has been detected by analysis of the dissociation constant in the presence of varying concentrations of neutral solutes and cosolvents. The dissociation constant as measured by gel mobility shift assays increased as the concentration of dimethyl sulfoxide, glycerol, sucrose, or polyethylene glycol was increased. Osmotic pressure is the only property that correlates with the change in the dissociation constant for all compounds. This data indicates that binding of a small population of water molecules accompanies formation of the Hin-DNA complex, and points to a novel role for solvent molecules in assisting site specific interaction between DNA-binding proteins and their cognate DNA sequence.

Understanding thermostability in cytochrome P450 by combinatorial mutagenesis.

The cytochromes P450 are an important class of mono-oxygenases involved in xenobiotic metabolism and steroid biosynthesis in a diverse set of life forms. Discovery of CYP-119, a P450 from the archea Sulfolobus solfataricus has provided a means for understanding nature's method of stabilizing this important protein superfamily. To identify classes of stabilizing interactions used by CYP-119, we have generated a randomized library of point mutants and screened for mutants that are less thermostable than the wild type by monitoring the characteristic Soret band in the visible region of the cell lysis. The selected mutants were characterized by differential scanning calorimetry to compare the temperatures of the melting transitions of the various mutants. The identified mutations suggested that electrostatic interactions involving salt links and charge-charge interactions, as well as contributions from other interactions such as aromatic stacking, and side chain volume of hydrophobic residues contribute to enhanced thermostability in this cytochrome P450.

Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3-hydroxynorcamphor product.

Cytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1-methyl-norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1-methyl-norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%, but for both norcamphor and 1-methyl-norcamphor, the efficiency is dramatically lowered to 12% and 50%, respectively. Based on an examination of the active site of P450cam, it appeared that mutating position 185 might dramatically alter the product specificity and coupling of hydroxylation of norcamphor by P450cam. Analysis of molecular dynamics trajectories of norcamphor bound to the T185F mutant of cytochrome P450cam predicted that hydroxylation at the 3 position should be abolished and that the coupling should be dramatically increased. This mutant was constructed and the product profile and coupling experimentally determined. The coupling was doubled, and hydroxylation at the 3 position was essentially abolished. Both of these results are in agreement with the prediction.

A differential scanning calorimetric study of the thermal unfolding of apo- and holo-cytochrome b562.

Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group. In the absence of heme, cytochrome b562 remains highly structured under native conditions. Here we report thermodynamic data for the thermal denaturation of the holo- and apoproteins as determined by differential scanning calorimetry. Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group. Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process. Apocytochrome b562 is substantially destabilized relative to the holoprotein: the t1/2 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values. However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins.

Development of broad-host-range vectors for expression of cloned genes in Pseudomonas.

The cloning and expression of genes in Pseudomonas have been difficult, until now, due to the absence of vector systems that contain multiple restriction sites downstream from promoter sequences that are functional in Pseudomonas. We report here the construction of several broad-host-range vectors that can be utilized in either Pseudomonas or Escherichia coli and that rely on easily selectable antibiotic resistance markers with multiple cloning sites. These vectors were constructed by inserting the entire pUC13 sequence into derivatives of the RSF1010 wide-host-range plasmid. From this construction, other derivatives were obtained, specifically a lacZ::KmR fusion gene which provides an easily selectable marker in both E. coli and Pseudomonas. These vectors have been used to express the Pseudomonas putida cytochrome P450 monoxygenase gene in a P450-deficient P. putida strain. Thus, these vectors allow for the cloning, expression and selection of Pseudomonas genes in Pseudomonas by complementation.

A critical role of protein-bound water in the catalytic cycle of cytochrome P-450 camphor.

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P-450cam were followed in the presence of co-solvents used to increase the osmotic pressure surrounding the protein-bound water. As a result, the measured Vmax decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of Vmax as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P-450cam-putidaredoxin interface.

Decreased substrate affinity upon alteration of the substrate-docking region in cytochrome P450(BM-3).

A mutation at the surface of the substrate access channel which dramatically decreases the affinity for some fatty acids in P450(BM-3) was discovered by random mutagenesis. The mutation introduced, proline-25 to glutamine, is in close proximity to the arginine-47 residue thought to be responsible for the initial docking of fatty acid substrates. The P25Q mutant displays an affinity for palmitate which is approximately 100-fold weaker than the wild-type enzyme. In addition to its altered substrate affinity, P25Q also exhibits altered hydroxylation specificity and carbon monoxide recombination kinetics in the substrate-free form.