Structural Dynamics of Myoglobin

C H A P T E R

T W E N T Y

Structural Dynamics of Myoglobin M. Brunori,* D. Bourgeois,† and B. Vallone* Contents 398 399 400 400 402 408 413 413

1. Background 2. Crystallographic Studies of Myoglobin States 3. Experimental Approaches 3.1. Crystallization and sample handling 3.2. Pump and probe picosecond Laue diffraction 3.3. Myoglobin relaxations: A synopsis Acknowledgments References

Abstract Protein structure is endowed with a complex dynamic nature, which rules function and controls activity. The experimental investigations that yield information on protein dynamics are carried out in solution; however, in most cases, the determination of protein structure is carried out by crystallography that relies on the diffraction properties of a large number of molecules, in approximately the same conformation, arranged in a three-dimensional lattice. Myoglobin, maybe the most thoroughly characterized protein, has allowed the formulation of general principles in the field of protein structure–function correlation and, since the late 1990s, it has been possible to obtain directly some insight into the complex dynamic behavior of myoglobin and other proteins by Laue diffraction. This chapter describes some of the technological features involved in obtaining reliable data by time-resolved Laue crystallography, with subnanosecond time resolution. A synopsis of the more significant findings obtained by laser photolysis of myoglobin-CO crystals is also presented, emphasizing the more general aspects of dynamics relevant to the complex energy landscape of a protein.

* {

Dipartimento di Scienze Biochimiche ‘‘A. Rossi Fanelli,’’ Universita` di Roma ‘‘La Sapienza,’’ Roma, Italy Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, Universite´ Joseph Fourier and European Synchrotron Radiation Facility, Grenoble Cedex, France

Methods in Enzymology, Volume 437 ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)37020-1

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2008 Elsevier Inc. All rights reserved.

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1. Background The essential role of dynamics in controlling protein activity is an accepted fact, based on a large number of experimental data indicating that the primary function, as well as the control of reactivity, has a dynamic component. These concepts were formulated, by and large, based on the interpretation of time-resolved spectroscopy and nuclear magnetic resonance. In this respect, myoglobin (Mb) acquired, over the years, the undisputed role of a paradigmatic case for studying the correlations between structure and dynamics; despite its small size and simple structure, Mb displays a dynamic behavior of considerable complexity and hence has been referred to as the ‘‘paradigm of complexity’’ (Frauenfelder et al., 2003). The discovery of geminate rebinding after laser photolysis of the oxygen-heme iron bond and the correlation of protein relaxation with overall rates of binding and dissociation were essential to set the stage. The combined use of time-resolved spectroscopy and site-directed mutagenesis yielded an overall kinetic picture of the binding of many photodissociable ligands (O2, CO, NO, and others) to ferrous Mb and an assessment of the role of the inner and outer barriers in controlling entry and escape of the ligand in the protein matrix (Scott et al., 2001). In addition, studies on the geminate recombination provided evidence for an unexpected role of the internal structure of the protein in controlling function. Far from being a compact homogeneous matrix, the protein interior contains packing defects and cavities, which were already identified in the mid-1970s (Richards, 1974). In the case of Mb, direct evidence for the presence of these cavities was obtained by crystallography under high pressures of xenon; Tilton et al. (1984) showed that met-Mb can bind four atoms of xenon in small preexisting cavities numbered from Xe1 to Xe4. The role of these cavities in Mb function has been assessed either by filling them with xenon or by mutagenesis (Brunori and Gibson, 2001; Scott et al., 2001). Many investigations contributed to our understanding of the general significance of protein dynamics and paved the way to the concept of a complex energy landscape of a protein. Frauenfelder et al. (1988, 1991) and Ansari et al. (1992) extensively analyzed the problem and highlighted the principal correlations between the function of a protein and its dynamics. Using time-resolved X-ray crystallographic methods to directly measure the relaxation of Mb over the whole range of times explored by the protein matrix is the focus of this chapter.

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2. Crystallographic Studies of Myoglobin States The notion that crystallography provides a static picture of the protein structure is widespread. Generally, understanding a mechanism demands analysis of the three-dimensional structure of a protein at least in the initial and final states of the reaction; seldom it proved possible to crystallize reaction intermediates and analogues. The dynamics of motions along the pathway from one state to another is not amenable to classical X-ray crystallography. Attempts to extract dynamic information from diffraction experiments have been published (Frauenfelder et al., 1979; Rasmussen et al., 1992). Frauenfelder et al. (1979) analyzed the asymmetric distribution of thermal B factors from diffraction data of a met-Mb crystal at different temperatures and attempted to correlate this distribution with the ligandlinked conformational changes. A more challenging and informative crystallographic approach to unveil the structure of reaction intermediates was based on using trapping techniques (Bourgeois and Royant, 2005). Mb proved especially suitable because of the well-known photosensitivity of the ligand-iron bond; thus, crystals of MbCO can be photodissociated by stationary light at ultralow temperatures (
10–20 K) so that intermediate states may accumulate in a steady-state regime. Analysis of the photolytic intermediate at ultralow temperatures (Schlichting et al., 1994; Teng et al., 1997) demonstrated, for the first time, that photodissociated CO resides in the distal heme pocket at a distance of 3.6 A˚ from the iron (reviewed by Schlichting and Chu, 2000). Using the same approach a few years later, Brunori et al. (2000) showed that the photodissociated ligand in the triple Mb mutant (called YQR) migrated away even at extremely low temperatures to populate the Xe4 cavity on the distal side. Informative as they are, these experiments failed to detect the larger motions of the protein moiety (stuck at ultralow temperatures) unless the crystal was progressively warmed up to the so-called dynamical transition temperature where ligand diffusion further away becomes detectable (Ostermann et al., 2000). These results showed how crucial it is to obtain time-resolved crystallographic data at room temperature (Bourgeois and Royant, 2005). This missing information was tackled by the pioneering work of Moffat and collaborators (Srajer et al., 1996); since the late 1990s, Laue diffraction results with nanosecond and subnanosecond time resolutions have provided considerable insight into the complex dynamic behavior of Mb and other proteins (Bourgeois et al., 2003; Genick et al., 1997; Ihee et al., 2005; Knapp et al., 2006; Schotte et al., 2003; Srajer et al., 2001). This chapter presents a short description of the methodological improvements that made it possible to crack this challenging technological tour de

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force, and presents a short synopsis of the essential results obtained by following the structural dynamics of Mb from picoseconds to milliseconds and the general conclusions emerging from this approach.

3. Experimental Approaches 3.1. Crystallization and sample handling The level of detail provided by recent time-resolved studies carried out on Mb-YQR (see later) is the result of both careful tuning of sample preparation and drastic improvements in the experimental strategy employed for the collection of pump-probe Laue data on specialized synchrotron beam lines. The quality of the samples is a mandatory prerequisite for the success of time-resolved experiments and can, to a certain extent, be assessed before data collection is attempted. Myoglobin crystals are normally grown in the oxidized ferric form (met-Mb) and are subsequently soaked with the appropriate ligand in order to analyze the bound state. In the case of carbon monoxide, the state competent for ligation is the ferrous one, and pretreatment with a reducing agent, usually sodium dithionite, is necessary in order to obtain the species competent for CO (and dioxygen) binding. This procedure is normally mild enough to yield samples that can still be subjected to data collection for standard structural studies. Nevertheless, in the case of Mb-YQR, reduction with dithionite and subsequent soaking with a CO-saturated mother liquor result in a substantial increase in mosaicity (from 0.3 to 0.7 and above) and in a loss of crystal order that hamper the detection of fine structural details and prevent the unambiguous detection of CO docking within protein cavities. This problem can be overcome by growing Mb-YQR crystals in batches starting from the ferrous CO-bound derivative; in this way, not only is crystal degradation upon treatment avoided but, at the same time, the contribution from residual ferric unbound protein, decreasing overall photolytic yield in the crystal, is essentially eliminated. A protocol describing the conditions for batch crystallization starting from a vapor diffusion setup and leading to growth of Mb-YQR-CO crystals suitable for timeresolved studies is provided in the following section. 3.1.1. Seed preparation Crystals are first grown using the vapor diffusion method (Phillips et al., 1990) in 2.7 M ammonium sulfate, 20 mM Tris-Cl, pH 8.7, 1 mM EDTA, and 20 mg/ml met-Mb-YQR. A single crystal with no apparent defect is crushed within 1 ml mother liquor, mixed with a vortex mixer for a few seconds, and serially diluted in mother liquor (from 1 to 10,000 times).

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These ‘‘seed stocks’’ have to be stored carefully sealed at 20 , and are effective for seeding purposes for a few months. 3.1.2. Search for optimal batch crystallization growth conditions First, determine the optimal seed dilution by setting up five batch crystallization trials in the following way: equilibrate distilled water, 4.1 M ammonium sulfate solution (A/S), 1.5 M Tris-Cl pH 8.7 (stock buffer), 0.1 M EDTA, and mineral oil with CO at 1 atm by bubbling under a gas hood; the CO concentration after equilibration in water at 20 is 1 mM (some deviations from this concentration are expected for the other solutions). Prepare anaerobically a solution of 15 mM sodium dithionite using the CO-equilibrated stock buffer. First lay 30 ml of mineral oil in each tube, add 10 ml of ferric Mb-YQR under the oil layer with a gas-tight microsyringe, and add 2 ml of dithionite 15 mM in CO-equilibrated stock buffer. This ensures that the protein is reduced and bound with CO in an anaerobic environment. Then add 0.2 ml of EDTA and water (if necessary) and the ammonium sulfate-saturated solution. Finally, mix the solution with a gastight microsyringe; some precipitation may appear. As a last step, add 0.5 ml of the five seed stock solutions at different dilutions, after mixing them thoroughly with a vortex mixer. The volumes of the different components to mix to obtain 0.1 M Tris-Cl, 1 mM EDTA, and 10 mg/ml Mb solutions of a final concentration of 2.8 M ammonium sulphate, are as follow: 5 ml protein at 40 mg/ml, 13.5 ml A/S, 1.3 ml of stock buffer plus 15 mM sodium dithionite, 0.2 ml of 0.1 M EDTA, and 0.5 ml of each seed stock solution. The Tris-Cl concentration is increased with respect to the vapor diffusion protocol given the presence of 10 mM dithionite; the protein concentration is decreased to reduce the amount necessary for setting up the experiment and, as a consequence, the precipitant (ammonium sulfate) concentration is increased slightly. Immediately after preparation, the tubes have to be stored at 20 in a glass vessel purged with CO. The seed dilution that is found to yield the optimal number of crystals (5–10 per vial) is 1:10. With that particular protein and seed batch using 2.8 M A/S and 5 mg/ml protein concentration, we obtain growth of crystals within 2–3 days, completed within 1 week with 5–10 single crystals in each tube of a size ranging from 100 to 400 mm in the largest dimension. Some precipitate is often observed in the, batches and crystals tend to grow at the oil–solution interface. Figure 20.1 shows an example of a microbatch vial after crystal growth. ˚ resolution in timeCrystals grown using this protocol diffract up to 1.55 A resolved experiments, with a mosaicity ranging between 0.25 and 0.30 (mosaicity is assessed independently by static X-ray data collection). They show 100% CO ligation and undetectable ferric Mb contamination, as assessed by microspectrophotometric measurements.

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Mineral oil

Gas tight wax Crystals

CO atmosphere

Oil/solution interface

Mineral oil Mineral oil

Mb crystal

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Figure 20.1 (Left) Microbatch-grown crystals of Mb-YQR-CO. Crystals grew at the oil/solution interface; some precipitate is present at the bottom of the vial. (Right) Crystal mounting of Mb-YQR-CO for time-resolved experiments.

Crystals are mounted in 1-mm quartz capillaries, using mother liquor equilibrated with CO and containing 1 mM sodium dithionite under 1 atm of CO. The presence of a reducing agent and gaseous CO ensures that Mb is kept reduced and bound to CO throughout the measurement. In order to reduce the effects of heat delivered onto the sample by laser illumination, mild cooling to 10  C is ensured by means of a nitrogen cryostream. The combination of laser heating and cryostream cooling tends to induce temperature gradients throughout the capillary, resulting in solvent distillation to/from the crystal. The problem can be overcome by mounting the crystal in the capillary between two plugs of mineral oil placed very close to the sample (see Fig. 20.1).

3.2. Pump and probe picosecond Laue diffraction Time-resolved Laue diffraction with subnanosecond resolution employs the ‘‘pump and probe’’ strategy: a laser pulse initiates the reaction within the crystal and is followed by an X-ray pulse providing diffraction data from the excited molecules (Schotte et al., 2004; Wulff et al., 2002). The X-ray pulse originates from a single electron bucket circulating around the synchrotron storage ring. The sequence needs to be repeated many times to (i) build up a sufficient signal-to-noise ratio for each individual diffraction pattern, (ii) collect diffraction patterns at several crystal orientations so as to sample the reciprocal space entirely, and (iii) obtain structural information at different pump-probe delays to scan the time dimension. Therefore, the

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reaction under study needs to relax back to the dark resting state in seconds or less so that many cycles can be performed on the same sample within a reasonable experimental time. The crystal must also withstand repeated laser and X-ray pulses without significant loss of diffraction quality. This is why the method is best suited for light-sensitive proteins that undergo reversible photo cycles and yield excellent quality and radiationhard crystals. Investigations of single-turnover reactions based, for example, on photolysis of caged compounds (Bourgeois et al., 2005) have been sometimes successful in the past (Schlichting et al.,1990; Stoddard et al., 1998), but seem poorly compatible with the now consensual ‘‘pink’’ Laue data collection strategy employed on most time-resolved beam lines (see later). 3.2.1. Crystal photolysis Reaction initiation by laser light within a protein crystal is one of the most challenging issues of time-resolved Laue experiments and is a critical determinant for success or failure. To provide efficient and homogeneous excitation throughout the sample, one must deal with the generally very high optical density of colored protein crystals. The laser wavelength, pulse energy, and pulse duration must also be chosen to minimize heating and temperature gradients across the crystal and to avoid spurious oxidation or ionization events. Laser pulses approaching the femtosecond regime may generate nonlinear or multiphotons effects that may pose insurmountable problems (P. Anfinrud, personal communication). Figure 20.2 shows a basic simulation of how light penetrates into a Mb-CO crystal at three different wavelengths. It is striking to see that photons at wavelengths near the Q absorption bands of the heme moiety penetrate very little into the crystal, whereas photons in the Soret band do not penetrate at all. Photons at remote wavelengths penetrate deeper but require much more laser power to achieve photolysis. In practice, Fig. 20.2 shows that a rather good compromise is obtained at
500 nm, provided that thin crystals (less than
50 mm thick) are considered. As Mb crystals are typically much thicker, either the X-ray focal spot must be restricted to match the illuminated layer of the crystal (solution adopted at beam line ID09-B of the ESRF) or the crystal must be illuminated from several sides to increase the excited volume (solution adopted at beam line 14-ID, BioCARS, Advanced Radiation Source at Argonne National Laboratory). With Mb-YQR crystals on beam line ID09-B of the ESRF, the X-ray beam (50 mm) is typically centered 25 mm below the illuminated top edge of the crystal. The simulations used in Fig. 20.2 predict an overall photolysis yield of 74% at 505 nm excitation, yet such a yield is never achieved. Many pitfalls combine to reduce the effective photolysis yield obtainable in crystals. Among these are imperfect alignment of the laser beam, X-ray beam, and crystal; loss of photolysis light as a consequence of Fresnel reflections on

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Figure 20.2 Theoretical penetration of visible laser light into a myoglobin crystal.The crystal is assumed to be composed of 80% Mb-CO and 20% of oxidized Mb-met. A laser pulse energy of 40 mJ is assumed that impinges on a crystal of dimensions 200  200  50 mm and space group P61. The calculation neglects effects discussed in the text, such as misalignments, polarization effects, Fresnel reflections, or transient change of absorption in the excited states.The calculation makes use of experimentally measured extinction coefficients of myoglobin in the CO-bound and oxidized met states, which are shown in the inset, together with those of the deoxy state. It is seen that whereas light at 582 nm (Q-band maximum) creates a large absorption gradient in the crystal, light at 630 nm is almost not absorbed. Light at
500 nm represents a good compromise, although only a maximum crystal thickness of
50 mm can be photolyzed successfully if only one laser beam is used.

capillary and crystal surfaces; and effect of nonrandom orientation of the absorbing groups within the crystal unit cell (potentially implying a dependence of photolysis yield on crystal orientation). In Mb, the presence of a fraction of oxidized met-Mb in the crystal, as well as incomplete CO saturation, will also alter the observed yield. In addition, other more fundamental aspects may play a role: the amount of very fast geminate recombination (on the subnanosecond timescale) might be enhanced in the crystalline state because of reduced conformational freedom or because of a different thermodynamic balance between conformational substates present in the crystal and in solution. At a given laser pulse energy, the photolysis yield might also depend on the pulse duration. When the pulse duration is shortened to the femtosecond regime, it may approach the lifetime of early excited states. Therefore, even if several photons are available per molecule, only the first one absorbed may lead to photolysis, as the following ones will then be dumped onto an already excited molecule that had no time to relax back to the ground state. Furthermore, the absorption spectrum of early excited states may strongly absorb at the excitation wavelength and transiently shield the crystal (P. Anfinrud, personal communication). On the contrary, longer laser pulses allow individual molecules to

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relax back to the ground state between successive absorption events, offering multiple chances for successful photolysis. In general, long pulse durations achieve better photolysis yield, while minimizing multiphoton excitations, and are therefore easier to handle. With pulses in the 100-ps to nanosecond regime, typically photolysis yields between 10 and 40% are obtained. On beam line ID09-B of the ESRF, laser pulses of
40 mJ energy, 100 ps duration, and with wavelengths spanning the 480- to 560-nm range have been used for studies on Mb. Pulses of 100 fs duration are produced by an optical parametric amplifier pumped by a Ti:sapphire regenerative amplifier. Temporal pulse stretching from 100 fs to 100 ps is realized by the combined use of a Brewster cut fused silica glass block and of an optical fiber transporting laser light to the focusing optics near the sample. The optical fiber also achieves spectral broadening so that a
40-nm band-pass spectrum impinges on the sample (Schotte et al., 2004). Under these conditions, in the case of Mb-YQR, which provides rather large crystals, it is usually possible to utilize a single crystal for collecting more than one set of time points, taking advantage of different regions of the crystal. The rather low laser energy employed also reduces the problem of crystal slippage, which was evident in early experiments when a hot laser spot delivering 1.0–1.5 mJ at softer wavelengths was used. In order to avoid movements of the crystal induced by laser flashes, a sticky polyvinyl coating can be adopted (Knapp et al., 2004). 3.2.2. X-ray data collection The collection of Laue data (Clifton et al., 1997; Cruickshank et al., 1991; Ren and Moffat, 1995a; Ren et al., 1999) with subnanosecond time resolution (Bourgeois et al., 1996; Wulff et al., 2002) has been reviewed in detail (Bourgeois et al., 2007) and is briefly reported. Time-resolved Laue data collection is carried out under specific configurations of the synchrotron ring: the so-called ‘‘single-bunch’’ or ‘‘few-bunches’’ modes are used, where one or few electron bunches generate very intense
100-ps X-ray pulses when traversing an insertion device. These X-ray pulses are now produced with narrow band-pass monoharmonic undulators, not with broad bandpass wigglers. Monoharmonic undulators deliver a hot, so-called ‘‘pink’’ beam, with most photons emitting in a single tooth-shaped line with a sharp energy cutoff. Development of the pink Laue concept followed from the realization that the broad X-ray band pass generated by wiggler insertion devices, despite large coverage of reciprocal space in a single shot, delivers data of rather poor quality (Bourgeois et al., 2000). Undulator beams with a few percent band pass improve the signal-to-noise ratio greatly because, while the intensity of each Laue diffraction spot is preserved, the X-ray noise at every pixel of the detector is reduced in proportion to the band pass.

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This improvement in the signal-to-noise ratio is at the expense of a reduced coverage of reciprocal space per frame, but this is a tractable problem for the study of cyclic reactions in crystals able to sustain repeated excitations. The sparse reciprocal lattice points stimulated by the pink beam also result in simplifications of the diffraction images, reducing the number of harmonic and spatial overlaps drastically (Ren et al., 1999), thus providing better raw data (Bourgeois et al., 2000). Enhanced evenness of reciprocal space coverage also squeezes the ‘‘low-resolution hole’’ typical of wiggler Laue diffraction data and known to cause discontinuities in electron density maps. Advantages in Laue geometry provided by monoharmonic undulators are further strengthened by a decrease in beam line heat load, resulting in a more stable beam. To allow for the selection of properly timed X-ray pulses on the crystal, high-speed choppers with opening times in the microsecond range have been designed. These devices rotate at about a kilohertz and are phase locked to the storage ring clock, ensuring synchronization to the X-ray beam with nanosecond mechanical jitter. Opening times down to
100 ns have been achieved. Synchronization between laser and X-ray pulses is realized by sophisticated electronics providing picosecond accuracy (Schotte et al., 2004). The collection of time-resolved Laue data occurs in a four-dimensional space and therefore should be planned carefully. To avoid systematic errors that may bias the observed structural changes along the time axis, e.g., because of crystal photo damage, time is used as the ‘‘fast variable.’’ At a given crystal orientation, all the diffraction images at the investigated pumpprobe delays (sampled equidistantly on the logarithmic timescale) are collected and then the crystal is rotated and the process is repeated (Ihee et al., 2005). A ‘‘laser off ’’ image is also recorded to provide a ‘‘dark state’’ reference structure of the sample. The calculation of difference electron density maps between different time points then mostly subtracts out irreversible structural changes caused by X-ray or laser light. It should be noted that such a data collection scheme spreads out errors along the time axis, but does not eliminate them. The quality of the diffraction images should therefore be checked throughout the experiment by visual inspection or preferably by online data processing. On-the-fly assessment of data quality during the course of the experiment is now possible thanks to recent progress in software and fast computers. In the last few years, methodology for reducing Laue data has improved considerably, providing accuracy and automation in addition to speed (F. Schotte, unpublished results; Bourgeois et al., 2000; Ren, 2006). Highly robust indexing algorithms have been developed, which are based on the recognition of entire conics and are therefore able to cope with the limited number of nodal spots present in ‘‘pink’’ Laue patterns (Ren, 2006 E. R. Henry, unpublished results). Wavelength-dependent prediction of

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the crystal resolution limit has been implemented and has brought about substantial improvement in data quality (Bourgeois et al., 2000). Spatial and harmonic overlaps are now deconvoluted computationally on a routine basis (Arzt et al., 1999; Bourgeois et al., 1998; Ren and Moffat, 1995b). However, other challenges remain in the processing of Laue data from narrow band-pass sources, as expected in the future from, for example, X-ray-free electron lasers. Proper wavelength normalization of ‘‘mosaic’’ or ‘‘partial’’ reflections is still lacking and will become of increasing importance as the X-ray band pass decreases. Structural changes at the investigated time delays are classically revealed by computing difference electron density Fourier maps preferably enhanced by Bayesian weighting of the (small) difference structure factor amplitudes (Ursby et al., 1997). Qualitative assessment of data may benefit from maps extrapolated to full photolysis to eliminate the contribution of the nonexcited fraction of the crystal, which can then be rendered with special coloring schemes so as to guide the eye in following the subtle motions of individual atoms (Schotte et al., 2004). Quantitative evaluation of data is often based on integrating density features in difference maps at the locations of interest so as to assess the evolution of their electron content over time (Bourgeois et al., 2003; Schmidt et al., 2005). The time evolution of these features may then be fitted by kinetic schemes (Knapp et al., 2006; Schmidt et al., 2005). However, the method suffers from a fundamental limitation, as it is the rule rather than the exception that several conformational states coexist in the crystal at any given time. Hence, density features generally do not represent a single species, but rather a combination of several species. This is why refinement of structural models from data at a single time point should be attempted and interpreted only with great care (Aranda et al., 2006; Bourgeois et al., 2003), preferably using the method of difference refinement (Terwilliger and Berendzen, 1995). To extract the structures of time-independent transient species, as well as their rates of interconversion, the technique of singular value decomposition (SVD) has been elegantly generalized to time-resolved crystallography (Rajagopal et al., 2004; Schmidt et al., 2003). This method works well when the amount of time points is sufficient (approximately three per decade) and when data are devoid of systematic errors along the time axis. In addition, SVD analysis provides an efficient noise filter and therefore enhances the signal-to-noise ratio in reconstituted electron density difference maps. However, kinetic models describing the time evolution of the investigated sample may only be shown to be consistent with data, i.e., they may not be proven to govern the reaction scheme. They may, though, be compared to the SVD outcome of spectroscopic data, which brings links between crystal and solution states and adds considerable weight to the validity of structural data (Yeremenko et al., 2005, 2006).

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3.3. Myoglobin relaxations: A synopsis The pioneer in this field is undoubtedly K. Moffat, who worked for two decades to obtain the first reliable time-resolved diffraction data presenting difference electron density maps between dark and photolyzed states of Mb (Srajer et al., 1996). The quality of results obtained at the time bears no comparison with current data due to progressive improvements in biochemistry, synchrotron technology and analytical methods, as outlined briefly earlier. Indeed, when looking at the paper by Srajer et al. (1996), it is clear that quantitative information was very limited; nevertheless this was a tremendous step forward, as it proved that the experiment was feasible and could potentially yield novel information. An intrinsic difficulty of the approach that will be hard to overcome is the yield of photodissociated Mb obtained by the laser flash, as well as the time window that can be explored before ligand recombination leads back to the equilibrium state. Over and above the problem related to laser penetration in the optically very dense crystal (see earlier discussion), it is obvious that ligand recombination events, either geminate or bimolecular, progressively reduce the yield of deoxy Mb photoproduct that undergoes relaxation. In this respect, site-directed mutagenesis proved very valuable; among others, the triple Mb mutant called YQR (Fig. 20.3) (Brunori et al., 1999) was found to be an excellent model system because mutated residues in the distal pocket were found to reduce geminate recombination to zero and to slow down (by 10-fold) the bimolecular recombination, extending

Tyr29 Gln64

Phe46

Haem

Bound CO Photolyzed CO (in Xe1 site) His93

Figure 20.3 Overview of the modeled structure of YQR-MbCO (dark gray) and YQR-Mb* (light gray). The heme and several key residues (Tyr29B10, Phe46CD4, Gln64E7, His93F8) are rendered as balls and sticks, the CO is rendered as space filling, and the rest of the protein is rendered as a ribbon. (From Bourgeois et al., 2003, modified).

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considerably the time window available for observation of the relaxation phenomena. Thus, it is very likely that engineering the globin in the future may produce additional variants that will allow the range of useful data acquisition to be extended. Wild-type sperm whale Mb and three mutants have been investigated in great detail (Bourgeois et al., 2003, 2006; Schmidt et al., 2005; Schotte et al., 2003; Srajer et al., 2001). In all cases, a fundamental feature of the time course after pholysis is the nonexponential extended relaxation of deoxy Mb, ranging from subnanoseconds up to milliseconds; in this respect, all the Mb variants tested share a common behavior. This first-order conclusion is very rewarding because it provides generality to the main observation. At the same time, subtle differences in dynamic behavior between mutants and the wild-type protein had to be interpreted and correlated with the extensive kinetics obtained in solution. Figure 20.3 and 20.4 depict a synopsis of data reported for Mb-YQR (Bourgeois et al., 2006). The outstanding result extracted from a quantitative analysis of the electron density difference Fourier maps is as follows: the relaxation profile of the heme and the globin moiety cover a time range from 150 ps (the earliest available time frame) to milliseconds when recombination to the dark state occurs. The latest data obtained on this triple mutant show that heme relaxation is at least biphasic, with some change occurring synchronously with the photolysis laser pulse, and additional shifts in heme geometry within nanoseconds. This relaxation behavior may also be present in the wild-type protein; however, in the specific case of Mb-YQR the heterogeneity was obvious and was attributed (Bourgeois et al., 2006) to a strain on heme pyrrole C exerted by the E helix via the CD turn. Most prominent from Fig. 20.4 is that the globin conformational change appears with a lag of several hundred nanoseconds, as may be seen by looking at the relaxation of the distal helix E and several residues of the CD turn. The extended relaxation behavior represents strong support to the idea (championed by Frauenfelder and colleagues) that photolysis sets into motion a protein quake; changes in the immediate environment of the heme, including distal side chains, occur rapidly, and the perturbation diffuses away with rearrangements of the globin at later times following the focal quake. The main results, which, despite some differences, are shared by essentially all the different variants, are of significance in the light of the energy landscape concept of a protein (Frauenfelder et al., 1991), which envisages an extended relaxation as the protein skates along a complex set of conformational coordinates. Moreover, the extended relaxation is the structural counterpart of numerous and detailed kinetic data obtained over the years by time-resolved spectroscopy (Ansari et al., 1994; Scott et al., 2001); as an example, we recall the photolytic experiments reported by Anfinrud and co-workers (Lim et al., 1997), which showed that the relaxation of

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Figure 20.4 Time dependence after photolysis of difference electron densities for key structural features. Numerical values reflect the integral of the positive electron density beyond 3.0s and are corrected for variations in photolysis yield.They are normalized so that the negative bound-CO feature is assigned a value of 1. Average values weighted by photolysis yield, over four independent data sets are shown. (A) Key features that appear

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photolyzed deoxy Mb covers a range from
10 ps up to many hundred microseconds. One more fact that is common and well established concerns the pathway of internal migration of the photolyzed CO, which remains trapped momentarily in the protein matrix, and the differences in the dynamics of these events among the different variants. The notion that CO migrates from the distal pocket of the heme through the protein matrix after photolysis was well anticipated by analysis of the geminate recombination profile of different ligands (CO, NO and O2) and by the effect of specific mutations and xenon at high pressures on the rebinding mechanism (Brunori and Gibson, 2001; Scott and Gibson, 1997; Scott et al., 2001). It was remarkable to confirm these hypotheses using Laue diffraction by watching directly the route followed by CO inside the protein. The permanence of CO in the distal pocket depends on the type of variant, being longer in the wild-type protein and considerably shorter with Mb-YQR (Bourgeois et al., 2006; Schotte et al., 2003); in the latter case, the photolyzed CO diffuses into the Xe4 cavity on the distal side almost immediately (<100 ps). This observation is in complete agreement with the lack of geminate recombination yield for Mb-YQR, and it was interpreted in terms of the different role played by the Gln-Tyr couple on the distal site, as compared to the wild-type His-Leu couple. The subsequent migration of CO along the web of cavities occurs at longer times (see Fig. 20.4) and displays essentially the same time course in the different variants. Thus the migration of CO from distal pocket to Xe4 and then to Xe2 and Xe1 on the proximal side takes around 100–300 ns, suggesting a correlation between ligand migration and large-scale structural relaxation of the globin; this point was analyzed carefully by Srajer et al. (2001) and compared with the recombination time course. This feature, which is well established, is not a peculiarity of Mb because it is known that internal packing defects identified in the structure of many proteins (especially from mesophiles) may represent temporary stations for small ligands or substrates entrapped inside the protein. Several authors have addressed the point of the possible significance of migration in the different cavities and functional control, as discussed in some review articles (Brunori et al., 2004; Lavalette et al., 2006; Nienhaus and Nienhaus, 2004; Teeter, 2004; Vallone and Brunori, 2004); some envisaged that the protein and its web of cavities may be viewed as a molecular reactor in more complex oxidative processes.

promptly. (B) Residues involved in the strain of the CD turn, lagging behind. (C) Population of CO in Xe1 and Xe4 sites and conformational changes of the E helix (where the average integrated density per residue is shown). (From Bourgeois et al., 2006, modified).

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A final important consideration afforded by recent data is the direct comparison between experimental results and molecular dynamics simulations. This has become possible because the time resolution of Laue diffraction data extended down to the hundreds of picoseconds while computational power allowed an increase of the length of the simulation up to
100 ns or above, providing an extended time overlap between the two techniques. A direct comparison of experimental data on protein relaxation and molecular dynamics simulations had not been possible before and it represents a test case of utmost importance. It is comforting that the two papers reporting a detailed comparison between experimental data and simulations (Bossa et al., 2005; Hummer et al., 2004) actually demonstrated an excellent agreement between the two approaches (Fig. 20.5); the opportunity to combine the two techniques is of tremendous significance for the future of the field (see Fig. 20.5). First of all, simulations may allow a clearer identification of essential dynamic features that may have escaped when DP

Xe4

Ph1

Ph2

Xe3 Xe2

Distance Å

Xe1

DP

6.0

Xe4

Ph1

Ph2

Xe3

Xe2

Xe1

4.0 2.0 0.0

0

10

20

70

80

90

Time (ns)

Figure 20.5 Crystal structure of the wild-type CO-bound sperm whale Mb is shown in the center. Cavities involved in the migration of the ligand, detected by the package SURFNET (Laskowski, 1995), are shown in dark gray and are indicated by legends; DP is the distal pocket. CO is not shown. (Bottom) The trajectory of the distance between the CO center of mass and the (arbitrarily chosen) center of each cavity is depicted. The different shades of gray and labels represent the locations of CO corresponding to cavities in the top panel. A few backward and forward transitions between adjacent cavities can be observed (DP-Xe4 at t ¼ 10 ns; Xe4-Ph1 at t ¼ 15 ns; Xe1-Xe2 in the 70- to 90-ns range); in the 25- to 65-ns time range (not shown), CO remains in the Xe1 cavity. It may be noticed that transitions in between cavities are very fast, showing that the ligand is hopping rapidly through channels connecting the cavities. (From Bossa et al., 2005, modified).

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looking at the ensemble averaged difference maps at different times. Moreover, the number of experimental time points along the much extended time course of relaxation of Mb is necessarily limited, contrary to the molecular dynamic trajectories. Last but not least, comparison to experimental data represents a control of validity of the procedures used in molecular dynamic simulations and thereby allows envisaging that these results may pave the way to a much more widespread application of this approach to describe the conformational relaxation of many proteins not amenable to Laue diffraction. After all, the approach by time-resolved Laue crystallography (informative as it is) is limited to heme proteins and a handful of other systems, given the absolute requirement to trigger a fully reversible process quickly and synchronously in the macromolecular crystal.

ACKNOWLEDGMENTS Work was partially supported by the Ministero Italiano dell’Universita` e della Ricerca (grants to M.B: FIRB 2003 RBLA03B3KC and PRIN 2005 ‘‘Dinamica strutturale di metalloproteine’’).

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