Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

Direct observation of ligand migration within human hemoglobin at work.

Hemoglobin is one of the best-characterized proteins with respect to structure and function, but the internal ligand diffusion pathways remain obscure and controversial. Here we captured the CO migration processes in the tense (T), relaxed (R), and second relaxed (R2) quaternary structures of human hemoglobin by crystallography using a high-repetition pulsed laser technique at cryogenic temperatures. We found that in each quaternary structure, the photodissociated CO molecules migrate along distinct pathways in the α and ß subunits by hopping between the internal cavities with correlated side chain motions of large nonpolar residues, such as α14Trp(A12), α105Leu(G12), ß15Trp(A12), and ß71Phe(E15). We also observe electron density evidence for the distal histidine [α58/ß63His(E7)] swing-out motion regardless of the quaternary structure, although less evident in α subunits than in ß subunits, suggesting that some CO molecules have escaped directly through the E7 gate. Remarkably, in T-state Fe(II)-Ni(II) hybrid hemoglobins in which either the α or ß subunits contain Ni(II) heme that cannot bind CO, the photodissociated CO molecules not only dock at the cavities in the original Fe(II) subunit, but also escape from the protein matrix and enter the cavities in the adjacent Ni(II) subunit even at 95 K, demonstrating the high gas permeability and porosity of the hemoglobin molecule. Our results provide a comprehensive picture of ligand movements in hemoglobin and highlight the relevance of cavities, nonpolar residues, and distal histidines in facilitating the ligand migration.

Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman, crystallography, and DFT calculation.

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or ß subunit contains a ferric heme in the α2ß2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.

X-ray fluorescence holography of biological metal sites: Application to myoglobin.

X-ray fluorescence holography (XFH) is a relatively new technique capable of providing unique three-dimensional structural information around specific atoms that act as a light source in crystalline samples. So far, XFH has typically been applied to inorganic materials such as dopants in metals and semiconductors. Here, we investigate the possibility of using XFH to visualize the metal active site in sperm whale myoglobin (Mb), a monomeric oxygen storage heme protein. We demonstrate that the atomic images reconstructed from the hologram data of crystals of carbonmonoxy myoglobin (MbCO) are moderately consistent with the crystal structure, which is also determined in this study by X-ray crystallography in the near-atomic resolution, as well as simulation results. These results open up a new avenue for the application of XFH to local atomic and electronic structure imaging of metal-sites in biomolecules.

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase.

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

Saturated Fatty Acids Undergo Intracellular Crystallization and Activate the NLRP3 Inflammasome in Macrophages.

OBJECTIVE: Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1ß (interleukin 1ß) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. APPROACH AND RESULTS: The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1ß release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1ß release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1ß release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1ß release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1ß release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1ß-deficient mice. CONCLUSIONS: These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Direct observation of conformational population shifts in crystalline human hemoglobin.

Although X-ray crystallography is the most commonly used technique for studying the molecular structure of proteins, it is not generally able to monitor the dynamic changes or global domain motions that often underlie allostery. These motions often prevent crystal growth or reduce crystal order. We have recently discovered a crystal form of human hemoglobin that contains three protein molecules allowed to express a full range of quaternary structures, whereas maintaining strong X-ray diffraction. Here we use this crystal form to investigate the effects of two allosteric effectors, phosphate and bezafibrate, by tracking the structures and functions of the three hemoglobin molecules following the addition of each effector. The X-ray analysis shows that the addition of either phosphate or bezafibrate not only induces conformational changes in a direction from a relaxed-state to a tense-state, but also within relaxed-state populations. The microspectrophotometric O2 equilibrium measurements on the crystals demonstrate that the binding of each effector energetically stabilizes the lowest affinity conformer more strongly than the intermediate affinity one, thereby reducing the O2 affinity of tense-state populations, and that the addition of bezafibrate causes an ∼5-fold decrease in the O2 affinity of relaxed-state populations. These results show that the allosteric pathway of hemoglobin involves shifts of populations rather than a unidirectional conversion of one quaternary structure to another, and that minor conformers of hemoglobin may have a disproportionate effect on the overall O2 affinity.

Delineation of solution burst-phase protein folding events by encapsulating the proteins in silica gels.

Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine ß-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.

Capturing the hemoglobin allosteric transition in a single crystal form.

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the half-liganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric O2 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.

The structural basis for an essential subunit interaction in influenza virus RNA polymerase.

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The carboxy-terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino-terminal residues of PB1 can fit by forming a 3(10) helix.

Structures and oxygen affinities of crystalline human hemoglobin C (ß6 Glu->Lys) in the R and R2 quaternary structures.

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (ß6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K(4)) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2-3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.

Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system.

We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 kinase inhibitors, and succeeded in screening two inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.

Tracking the Structural Development of Amyloid Precursors in the Insulin B Chain and the Inhibition Effect by Fibrinogen.

Amyloid fibrils are abnormal protein aggregates associated with several amyloidoses and neurodegenerative diseases. Prefibrillar intermediates, which emerge before amyloid fibril formation, play an important role in structure formation. Therefore, to prevent fibril formation, the mechanisms underpinning the structural development of prefibrillar intermediates must be elucidated. An insulin-derived peptide, the insulin B chain, is known for its stable accumulation of prefibrillar intermediates. In this study, the structural development of B chain prefibrillar intermediates and their inhibition by fibrinogen (Fg) were monitored by transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) combined with solid-state nuclear magnetic resonance spectroscopy (NMR) and size exclusion chromatography. TEM images obtained in a time-lapse manner demonstrated that prefibrillar intermediates were wavy rod-like structures emerging from initial non-rod-like aggregates, and their bundling was responsible for protofilament formation. Time-resolved SAXS revealed that the prefibrillar intermediates became thicker and longer as a function of time. Solid-state NMR measurement suggested a ß-sheet formation around Ala14 residue was crucial for the structural conversion from prefibrillar intermediates to amyloid fibril. These observations suggested that prefibrillar intermediates serve as reaction fields for amyloid nucleation and its structural propagation. Time-resolved SAXS also demonstrated that Fg prevented elongation of the prefibrillar intermediates by forming specific complexes together, which implied that regulation of the length of prefibrillar intermediates upon Fg binding was the factor suppressing the prefibrillar intermediate elongation. The fibril formation mechanism and the inhibition strategy found in this study will be helpful in seeking appropriate methods against amyloid-related diseases.

Freezable and Unfreezable Hydration Water: Distinct Contributions to Protein Dynamics Revealed by Neutron Scattering.

Hydration water plays a crucial role for activating the protein dynamics required for functional expression. Yet, the details are not understood about how hydration water couples with protein dynamics. A temperature hysteresis of the ice formation of hydration water is a key phenomenon to understand which type of hydration water, unfreezable or freezable hydration water, is crucial for the activation of protein dynamics. Using neutron scattering, we observed a temperature-hysteresis phenomenon in the diffraction peaks of the ice of freezable hydration water, whereas protein dynamics did not show any temperature hysteresis. These results show that the protein dynamics is not coupled with freezable hydration water dynamics, and unfreezable hydration water is essential for the activation of protein dynamics. Decoupling of the dynamics between unfreezable and freezable hydration water could be the cause of the distinct contributions of hydration water to protein dynamics.

Protonation states of histidine and other key residues in deoxy normal human adult hemoglobin by neutron protein crystallography.

The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F(o)-F(c) and 2F(o)-F(c) neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or ß chains, αHis20, αHis50, αHis58, αHis89, ßHis63, ßHis143 and ßHis146, have different protonation states. The protonation of distal His residues in the α(1)ß(1) heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK(a) between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.

Allosteric transitions in hemoglobin revisited.

BACKGROUND: Human hemoglobin is an allosteric protein that exerts exquisite control over ligand binding through large-scale conformational changes. The two-state model without intermediates offers a simple qualitative description of the allosteric behavior of hemoglobin, as presented in textbooks. However, there is renewed interest in this topic due to recent experimental breakthroughs that show how hemoglobin actually undergoes conformational transitions in response to environmental changes. SCOPE OF REVIEW: I review the current understanding of hemoglobin structure-function relationships revealed by recent discoveries. A unique single crystal, in which three protein molecules are allowed to express a whole range of quaternary structures, helped to reveal the detailed transition pathway including various intermediate forms. I also discuss the potential of single-molecule techniques that are currently under examination. MAJOR CONCLUSIONS: New crystallographic approaches reveal that the hemoglobin allosteric transition involves population shifts in multiple quaternary conformers rather than a simple two-state switch, and that coexisting individual conformers may have disproportionate effects on the apparent O2 affinity of hemoglobin. GENERAL SIGNIFICANCE: These approaches provide a further level of complexity on the textbook statement of hemoglobin allostery, highlighting the relevance of conformational distributions in controlling the function and regulation of allosteric proteins.

Pumping mechanism of NM-R3, a light-driven bacterial chloride importer in the rhodopsin family.

A newly identified microbial rhodopsin, NM-R3, from the marine flavobacterium Nonlabens marinus, was recently shown to drive chloride ion uptake, extending our understanding of the diversity of mechanisms for biological energy conversion. To clarify the mechanism underlying its function, we characterized the crystal structures of NM-R3 in both the dark state and early intermediate photoexcited states produced by laser pulses of different intensities and temperatures. The displacement of chloride ions at five different locations in the model reflected the detailed anion-conduction pathway, and the activity-related key residues-Cys105, Ser60, Gln224, and Phe90-were identified by mutation assays and spectroscopy. Comparisons with other proteins, including a closely related outward sodium ion pump, revealed key motifs and provided structural insights into light-driven ion transport across membranes by the NQ subfamily of rhodopsins. Unexpectedly, the response of the retinal in NM-R3 to photostimulation appears to be substantially different from that seen in bacteriorhodopsin.

Slow motion analysis of protein folding intermediates within wet silica gels.

Resolving the complete folding pathway of a protein is a major challenge to conventional experimental methods because of the rapidity and complexity of folding. Here, we show that entrapment of the protein cytochrome c in wet, optically transparent, porous silica gel matrices has enabled a dramatic expansion, to days or weeks, of the folding time, allowing direct observation of the entire folding pathway using a combination of three spectroscopic techniques, far-ultraviolet circular dichroism, tryptophan fluorescence, and Soret absorption spectroscopy. During refolding in silica gels, collapse and helix formation occur in a stepwise manner, as observed in aqueous solution. Analysis of kinetics and transient spectra indeed reveals a sequence of four distinct intermediates with progressively increasing degrees of folding, two of which closely resemble those previously characterized in solution, namely, the early collapsed and the molten globule intermediates. The other two are the precollapsed and pre-molten globule intermediates that may escape detection by conventional kinetic methods. Interestingly, varying the strength of the gel network has a dramatic effect on the folding time, but no significant effect on the structural features of each folding intermediate, indicating that the interaction between the protein and gel matrix has no measurable effect on the folding pathway. These results better define the major pathway of cytochrome c folding. In addition, in this paper we present the results of the application of this method to a simple, apparent two-state folder ubiquitin, helping to interpret the results for cytochrome c.

Circular dichroism study on the early folding events of beta-lactoglobulin entrapped in wet silica gels.

beta-Lactoglobulin is a predominantly beta-sheet protein that folds by forming excess alpha-helices within milliseconds. In this study, the refolding of beta-lactoglobulin was dramatically decelerated by entrapping in wet nanoporous silica gel matrices, and monitored on a time scale of minutes or hours by far-UV circular dichroism spectroscopy. Analysis of kinetics and transient spectra allowed to define the sequence of folding events that consist of alpha-helical formation, beta-sheet core formation, and alpha-to-beta transition. The results suggest that the initially formed alpha-helices, presumably including the native alpha-helix, help to guide the formation of the adjacent beta-sheet core.