Shigeru Matsuoka

A three-dimensional movie of structural changes in bacteriorhodopsin

Snapshots of bacteriorhodopsin Bacteriorhodopsin is a membrane protein that harvests the energy content from light to transport protons out of the cell against a transmembrane potential. Nango et al. used timeresolved serial femtosecond crystallography at an x-ray free electron laser to provide 13 structural snapshots of the conformational changes that occur in the nanoseconds to milliseconds after photoactivation. These changes begin at the active site, propagate toward the extracellular side of the protein, and mediate internal protonation exchanges that achieve proton transport. Science, this issue p. 1552 Time-resolved serial crystallography using an x-ray free electron laser reveals structural changes in bacteriorhodopsin. Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.

Acetate labeling patterns of dinoflagellate polyketides, amphidinols 2, 3 and 4

Abstract Amphidinols, which are polyketide metabolites chiefly comprising a long linear chain with polyhydroxyl groups and polyolefins, are produced by marine dinoflagellates Amphidinium carterae and A. klebsii. The acetate incorporation experiments of amphidinols 2, 3 and 4 revealed that they are built up with five regular C2-elongation sequences, which are separated by continuous acetate-methyl derived carbons. The findings support the C1-deletion mechanism from the regular sequence, which could be accounted for either by Favorski-type reaction or by Tiffeneau–Demjanov rearrengement.

Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B

It is widely accepted that amphotericin B (AmB) together with sterol makes a mixed molecular assemblage in phospholipid membrane. By adding AmB to lipids prior to preparation of large unilamellar vesicles (LUV), we directly measured the effect of cholesterol on assemblage formation by AmB without a step of drugs binding to phospholipid bilayers. Potassium ion flux assays based on 31P-nuclear magnetic resonance (NMR) clearly demonstrated that cholesterol markedly inhibits ion permeability induced by membrane-bound AmB. This could be accounted for by a membrane-thickening effect of cholesterol since AmB actions are known to be markedly affected by the thickness of membrane. Upon addition of AmB to an LUV suspension, the ion flux gradually increased with increasing molar ratios of cholesterol up to 20 mol%. These biphasic effects of cholesterol could be accounted for, at least in part, by the ordering effect of cholesterol.

Water-Mediated Recognition of Simple Alkyl Chains by Heart-Type Fatty-Acid-Binding Protein†

Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart-type FABP (FABP3) preferentially incorporates a U-shaped FA of C10–C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.

Amphotericin B–phospholipid covalent conjugates: dependence of membrane-permeabilizing activity on acyl-chain length

The interaction between amphotericin B and phospholipid upon forming ion channels across a biomembrane was investigated using their covalent conjugates. The membrane permeabilizing activity was greatly affected by the chain length of the fatty acyl groups, suggesting that their interaction is involved in ion channel assemblages.

Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid

The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15u2005Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate.

Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent

Significance This study shows successful experimental phasing methods (single-wavelength anomalous diffraction, single isomorphous replacement, and single isomorphous replacement with anomalous scattering) for crystal structure determination of a membrane protein by serial femtosecond crystallography with X-ray free electron lasers. Our iodine-containing detergent provided strong anomalous and isomorphous difference signals, which enabled experimental phasing using lower-resolution reflections (worse than 3 Å) from fewer indexed images than phasing attempts reported previously. The findings of this study will be applicable to a wide range of target proteins in structural biology, especially membrane proteins that often diffract to low resolution. The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 Å) X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1-Å resolution from only 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-Å resolution, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.

Novel Raman-tagged sphingomyelin that closely mimics original raft-forming behavior

Three Raman probes of sphingomyelin (SM) were synthesized and evaluated for their applicability to imaging experiments. One probe containing a hydroxymethyl-1,3-butadiyne moiety in the polar head group showed strong scattering. The solid-state (2)H NMR spectra of this probe in oriented bilayer membrane revealed excellent compatibility with natural SM in phase behavior since the probe undergoes phase separation to form raft-like liquid ordered (Lo) domains in the raft-mimicking mixed bilayers.

Molecular Dynamics Simulations of Heart-type Fatty Acid Binding Protein in Apo and Holo Forms, and Hydration Structure Analyses in the Binding Cavity

Intracellular lipid binding proteins (iLBPs) share distinctive features: a rigid protein structure composed of a β-barrel and an α-helix cap, and a large internalized water cluster. Although X-ray crystallographic studies have elucidated the three-dimensional structures of iLBPs, the protein dynamics and the role of the large water cluster in protein function remain unknown. In the present study, we performed molecular dynamics (MD) simulations on human heart-type fatty acid binding protein (FABP3), a typical iLBP that is highly expressed in heart and skeletal muscles, and showed that an altered mode of protein dynamics and rearrangement of the internal water cluster are key elements of ligand binding. Using simulations without a ligand at 310 K, we first demonstrated that FABP3 adopts a wide-open conformation, achieved by a combination of two modes of dynamics: portal opening by a domain motion of the α-helices and gap opening by cleavage of the hydrogen-bond network between βD and βE strands. In contrast, stearic acid-bound FABP3 mainly adopted a closed form, stabilized by the H-bond network inside the binding cavity, which latches the gap, and by protein-ligand hydrophobic interactions. The wide-open apo FABP3 represents a biologically important conformation relevant to ligand loading.

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry-Based Approach.

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their structure does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology.