ABSTRACT
Understanding protein structure and kinetics under physiological conditions is crucial for elucidating complex biological processes. While time-resolved (TR) techniques have advanced to track molecular actions, their practical application in biological reactions is often confined to reversible photoreactions within limited experimental parameters due to inefficient sample utilization and inflexibility of experimental setups. Here, we introduce serial X-ray liquidography (SXL), a technique that combines time-resolved X-ray liquidography with a fixed target of serially arranged microchambers. SXL breaks through the previously mentioned barriers, enabling microgram-scale TR studies of both irreversible and reversible reactions of even a non-photoactive protein. We demonstrate its versatility in studying a wide range of biological reactions, highlighting its potential as a flexible and multi-dimensional assay framework for kinetic and structural characterization. Leveraging X-ray free-electron lasers and micro-focused X-ray pulses promises further enhancements in both temporal resolution and minimizing sample quantity. SXL offers unprecedented insights into the structural and kinetic landscapes of molecular actions, paving the way for a deeper understanding of complex biological processes.
Subject(s)
Proteins , Kinetics , X-Rays , Proteins/chemistry , Protein Conformation , Lasers , Bacterial Proteins/chemistry , Bacterial Proteins/metabolismABSTRACT
Crystalline systems consisting of small-molecule building blocks have emerged as promising materials with diverse applications. It is of great importance to characterize not only their static structures but also the conversion of their structures in response to external stimuli. Femtosecond time-resolved crystallography has the potential to probe the real-time dynamics of structural transitions, but, thus far, this has not been realized for chemical reactions in non-biological crystals. In this study, we applied time-resolved serial femtosecond crystallography (TR-SFX), a powerful technique for visualizing protein structural dynamics, to a metal-organic framework, consisting of Fe porphyrins and hexazirconium nodes, and elucidated its structural dynamics. The time-resolved electron density maps derived from the TR-SFX data unveil trifurcating structural pathways: coherent oscillatory movements of Zr and Fe atoms, a transient structure with the Fe porphyrins and Zr6 nodes undergoing doming and disordering movements, respectively, and a vibrationally hot structure with isotropic structural disorder. These findings demonstrate the feasibility of using TR-SFX to study chemical systems.
ABSTRACT
Photoactive yellow protein (PYP) is one of the most extensively studied photoreceptors. Nevertheless, the role of the N-terminus in the photocycle and structural transitions is still elusive. Here, we attached additional amino acids to the N-terminus of PYP and investigated the effect of the length and charge of additional N-terminal residues using circular dichroism, two-dimensional nuclear magnetic resonance (2D-NMR), transient absorption (TA), and transient grating (TG) spectroscopic techniques. TA experiments showed that, except for negatively charged residues (5D-PYP), additional N-terminal residues of PYP generally enable faster dark recovery from the putative signaling state (pB2) to the ground state (pG). TG data showed that although the degree of structural changes can be controlled by adjusting specific amino acid residues in the extended N-terminus of N-terminal extended PYPs (NE-PYPs), the dark recovery times of wt-PYP and NE-PYPs, except for 5D-PYP, are independent of the structural differences between pG and pB2 states. These results demonstrate that the recovery time and the degree of structural change can be regulated by controlling the length and sequence of N-terminal residues of PYP. The findings in this study emphasize the need for careful attention to the remaining amino acid residues when designing recombinant proteins for genetic engineering purposes.
Subject(s)
Bacterial Proteins , Photoreceptors, Microbial , Bacterial Proteins/chemistry , Photoreceptors, Microbial/chemistry , Recombinant Proteins/chemistry , Circular Dichroism , Amino AcidsABSTRACT
Obtaining the heterogeneous conformation of small proteins is important for understanding their biological role, but it is still challenging. Here, we developed a multi-tilt nanoparticle-aided cryo-electron microscopy sampling (MT-NACS) technique that enables the observation of heterogeneous conformations of small proteins and applied it to calmodulin. By imaging the proteins labeled by two gold nanoparticles at multiple tilt angles and analyzing the projected positions of the nanoparticles, the distributions of 3D interparticle distances were obtained. From the measured distance distributions, the conformational changes associated with Ca2+ binding and salt concentration were determined. MT-NACS was also used to track the structural change accompanied by the interaction between amyloid-beta and calmodulin, which has never been observed experimentally. This work offers an alternative platform for studying the functional flexibility of small proteins.
Subject(s)
Calmodulin , Metal Nanoparticles , Cryoelectron Microscopy/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Protein ConformationABSTRACT
A fundamental problem in biological sciences is understanding how macromolecular machines work and how the structural changes of a molecule are connected to its function. Time-resolved techniques are vital in this regard and essential for understanding the structural dynamics of biomolecules. Time-resolved small- and wide-angle X-ray solution scattering has the capability to provide a multitude of information about the kinetics and global structural changes of molecules under their physiological conditions. However, standard protocols for such time-resolved measurements often require significant amounts of sample, which frequently render time-resolved measurements impossible. A cytometry-type sheath co-flow cell, developed at the BioCARS 14-ID beamline at the Advanced Photon Source, USA, allows time-resolved pump-probe X-ray solution scattering measurements to be conducted with sample consumption reduced by more than ten times compared with standard sample cells and protocols. The comparative capabilities of the standard and co-flow experimental setups were demonstrated by studying time-resolved signals in photoactive yellow protein.
Subject(s)
Proteins , Synchrotrons , X-Rays , Proteins/chemistry , Radiography , Photons , X-Ray DiffractionABSTRACT
A salt bridge, one of the representative structural factors established by non-covalent interactions, plays a crucial role in stabilizing the structure and regulating the protein function, but its role in dynamic processes has been elusive. Here, to scrutinize the structural and functional roles of the salt bridge in the process of performing the protein function, we investigated the effects of salt bridges on the allosteric structural transition of homodimeric hemoglobin (HbI) by applying time-resolved X-ray solution scattering (TRXSS) to the K30D mutant, in which the interfacial salt bridges of the wild type (WT) are abolished. The TRXSS data of K30D are consistent with the kinetic model that requires one monomer intermediate in addition to three structurally distinct dimer intermediates (I1, I2, and I3) observed in WT and other mutants. The kinetic and structural analyses show that K30D has an accelerated biphasic transition from I2 to I3 by more than nine times compared to WT and lacks significant structural changes in the transition from R-like I2 to T-like I3 observed in WT, unveiling that the loss of the salt bridges interrupts the R-T allosteric transition of HbI. Besides, the correlation between the bimolecular CO recombination rates in K30D, WT, and other mutants reveals that the bimolecular CO recombination is abnormally decelerated in K30D, indicating that the salt bridges also affect the cooperative ligand binding in HbI. These comparisons of the structural dynamics and kinetics of K30D and WT show that the interfacial salt bridges not only assist the physical connection of two subunits but also play a critical role in the global structural signal transduction of one subunit to the other subunit via a series of well-organized structural transitions.
ABSTRACT
Ultrafast motion of molecules, particularly the coherent motion, has been intensively investigated as a key factor guiding the reaction pathways. Recently, X-ray free-electron lasers (XFELs) have been utilized to elucidate the ultrafast motion of molecules. However, the studies on proteins using XFELs have been typically limited to the crystalline phase, and proteins in solution have rarely been investigated. Here we applied femtosecond time-resolved X-ray solution scattering (fs-TRXSS) and a structure refinement method to visualize the ultrafast motion of a protein. We succeeded in revealing detailed ultrafast structural changes of homodimeric hemoglobin involving the coherent motion. In addition to the motion of the protein itself, the time-dependent change of electron density of the hydration shell was tracked. Besides, the analysis on the fs-TRXSS data of myoglobin allows for observing the effect of the oligomeric state on the ultrafast coherent motion.
Subject(s)
Hemoglobins/chemistry , Kinetics , Molecular Dynamics Simulation , Myoglobin/chemistry , Protein Conformation , Protein Multimerization , Solutions , X-Ray DiffractionABSTRACT
Polymerase η (Polη) is one of the Y-family polymerases that is recruited by monoubiquitinated proliferating cell nuclear antigen (Ub-PCNA) to DNA damage sites during translesion synthesis (TLS). This interaction is mediated by an ubiquitin-binding zinc-finger (UBZ) domain and a PCNA-interacting protein (PIP) box in Polη, which binds to ubiquitin and PCNA, respectively. Here, we show that without the UBZ domain, the PIP box of yeast Polη has a novel binding function with ubiquitin. Furthermore, the UBZ domain and the PIP box share the same binding surfaces for ubiquitin. The interaction with ubiquitin via the PIP box stabilizes the Ub-PCNA/Polη complex. Moreover, the PIP residues I624 and L625 contribute to Polη function in TLS in vivo.
Subject(s)
DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Amino Acid Sequence , DNA/biosynthesis , DNA Damage , DNA Replication , Isoleucine/metabolism , Leucine/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Proliferating Cell Nuclear Antigen/metabolism , Protein Binding , Protein Domains , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Zinc FingersABSTRACT
Photoactive yellow protein (PYP) induces negative phototaxis in Halorhodospira halophila via photoactivation triggered by light-mediated chromophore isomerization. Chromophore isomerization proceeds via a volume-conserving isomerization mechanism due to the hydrogen-bond network and steric constraints inside the protein, and causes significant conformational changes accompanied by N-terminal protrusion. However, it is unclear how the structural change of the chromophore affects the remote N-terminal domain. To understand photocycle-related structural changes, we investigated the structural aspect of chromophore removal in PYP because it possesses a disrupted hydrogen-bond network similar to that in photocycle intermediates. A comparison of the structural aspects with those observed in the photocycle would give a clue related to the structural change mechanism in the photocycle. Chromophore removal effects were assessed via UV-vis spectroscopy, circular dichroism, and X-ray solution scattering. Molecular shape reconstruction and an experiment-restrained rigid-body molecular dynamics simulation based on the scattering data were performed to determine protein shape, size, and conformational changes upon PYP bleaching. Data show that chromophore removal disrupted the holo-PYP structure, resulting in a small N-terminal protrusion, but the extent of conformational changes was markedly less than those in the photocycle. This indicates that disruption of the hydrogen-bond network alone in bleached PYP does not induce the large conformational change observed in the photocycle, which thus must result from the organized structural transition around the chromophore triggered by chromophore photoisomerization along with disruption of the hydrogen-bond network between the chromophore and the PYP core.
Subject(s)
Bacterial Proteins/chemistry , Photoreceptors, Microbial/chemistry , Circular Dichroism , Molecular Dynamics Simulation , Protein Conformation , Scattering, Small Angle , Spectrophotometry, Ultraviolet , X-Ray DiffractionABSTRACT
Using various spectroscopic techniques such as UV-visible spectroscopy, circular dichroism spectroscopy, NMR spectroscopy, small-angle X-ray scattering, transient grating, and transient absorption techniques, we investigated how cell-mimetic environments made by crowding influence the photocycle of photoactive yellow protein (PYP) in terms of the molecular volume change and kinetics. Upon addition of molecular crowding agents, the ratio of the diffusion coefficient of the blue-shifted intermediate (pB) to that of the ground species (pG) significantly changes from 0.92 and approaches 1.0. This result indicates that the molecular volume change accompanied by the photocycle of PYP in molecularly crowded environments is much smaller than that which occurs in vitro and that the pB intermediate under crowded environments favors a compact conformation due to the excluded volume effect. The kinetics of the photocycle of PYP in cell-mimetic environments is greatly decelerated by the dehydration, owing to the interaction between the protein and small crowding agents, but is barely affected by the excluded volume effect. The results lead to the inference that the signaling transducer of PYP may not necessarily utilize the conformational change of PYP to sense the signaling state.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Halorhodospira halophila/metabolism , Photoreceptors, Microbial/chemistry , Photoreceptors, Microbial/metabolism , Biomimetics , Halorhodospira halophila/chemistry , Kinetics , Photochemical ProcessesABSTRACT
Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3',3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.
Subject(s)
Bacillus subtilis/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Dinucleoside Phosphates/metabolism , Staphylococcus aureus/chemistry , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Cation Transport Proteins/genetics , Dinucleoside Phosphates/chemistry , Models, Molecular , Potassium/metabolism , Protein Structure, Tertiary , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolismSubject(s)
DNA Helicases/chemistry , DNA Helicases/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Amino Acid Motifs , Amino Acid Sequence , Calorimetry , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Proliferating Cell Nuclear Antigen/ultrastructure , Protein Binding , Structural Homology, Protein , Structure-Activity RelationshipABSTRACT
OBJECTIVES: As the disabled have higher prevalence rates and earlier onsets of chronic diseases than the non-disabled, their participation in mass screening is important for the early detection and intervention of chronic diseases. Nevertheless, in Korea, the disabled have lower participation rates in mass screening services than the non-disabled. The purpose of the study was to find determinants for the participation in the National Health Insurance (NHI) mass screening program among the disabled. METHODS: In this study, the NHI mass screening data of 423,076 disabled people, which were identified using the National Disability Registry (2003), were analyzed. Of the factors affecting the participation rates in mass screenings, the following variables were included for the analysis: socioeconomic stati, such as sex, age, category of health insurance program, region and income; disability characteristics, such as disability type, and severity. A multiple logistic regression analysis was used to evaluate the association between the participation rates, disability characteristics variables and demographic variables. RESULTS: The participation rate in mass screening of the disabled was 41.3%, but was lower in females, an age of more than 70 years, self-employed and for those with an average monthly insurance premium over 133,500 Won and in metropolitan regions. The participation rate was 1.31 times lower in females than males (95% CI=1.29-1.33); 3.50 times lower in the elderly (more than 70 years) than the younger (95% CI=3.33-3.67); 1.43 times lower in those who live in metropolitan areas (95% CI=1.40-1.46); 2.59 times lower for those in a health insurance program for the self-employed than for employees (95% CI=2.56-2.63); 1.19 times lower for the higher income (more than 133,500) than the lower income group (4,400-22,000) for the average monthly insurance premium (95% CI=1.15-1.23); 2.04 times lower for those with brain palsy and stroke disabilities than with auditory impairments (95% CI= 1.97-2.11) and 3.27 times for those with severe compared to mild disabilities (95% CI=3.15-3.40). CONCLUSIONS: The disabled with high severity, and locomotive and communication disabilities have lower participation rates in mass screening services in Korea.