Kyeong Sik Jin

pH-dependent structures of ferritin and apoferritin in solution: disassembly and reassembly.

The pH-dependent structures of the ferritin shell (apoferritin, 24-mer) and the ferrihydrite core, under physiological conditions that permit enzymatic activity, were investigated by synchrotron small-angle X-ray scattering (SAXS). The solution structure of apoferritin was found to be nearly identical to the crystal structure. The shell thickness and hollow core volumes were estimated. The intact hollow spherical apoferritin was stable over a wide pH range, 3.40-10.0, and the ferrihydrite core was stable over the pH range 2.10-10.0. The apoferritin subunits underwent aggregation below pH 0.80, whereas the ferrihydrite cores aggregated below pH 2.10 as a result of the disassembly of the ferritin shell under the strongly acidic conditions. As the pH decreased from 3.40 to 0.80, apoferritin underwent stepwise disassembly by first forming a hollow sphere with two holes, then a headset-shaped structure, and, finally, rodlike oligomers. As the pH was increased from pH 1.96, the disassembled rodlike oligomers recovered only to the headset-shaped structure, and the disassembled headset-shaped intermediates recovered only to the hollow spherical structure with two hole defects. The apoferritin hole defects that formed during the disassembly process did not heal as the pH was increased to neutral or slightly basic conditions. The pH-induced apoferritin disassembly and reassembly processes were not fully reversible, although they were pseudoreversible over a limited pH range, between 10.0 and 2.66.

Molecular Basis for SMC Rod Formation and Its Dissolution upon DNA Binding

Summary SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive “hinge” dimerization domain with an ATP-regulated “head” dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB’s association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc’s dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome.

Small-angle x-ray scattering station 4C2 BL of pohang accelerator laboratory for advance in Korean polymer science

There are two beamlines (BLs), 4C1 and 4C2, at the Pohang Accelerator Laboratory that are dedicated to small angle X-ray scattering (SAXS). The 4C1 BL was constructed in early 2000 and is open to public users, including both domestic and foreign researchers. In 2003, construction of the second SAXS BL, 4C2, was complete and commissioning and user support were started. The 4C2 BL uses the same bending magnet as its light source as the 4C1 BL. The 4C1 BL uses a synthetic double multilayer monochromator, whereas the 4C2 BL uses a Si(111) double crystal monochromator for both small angle and wide angle X-ray scattering. In the 4C2 BL, the collimating mirror is positioned behind the monochromator in order to enhance the beam flux and energy resolution. A toroidal focusing mirror is positioned in front of the monochromator to increase the beam flux and eliminate higher harmonics. The 4C2 BL also contains a digital cooled charge coupled detector, which has a wide dynamic range and good sensitivity to weak scattering, thereby making it suitable for a range of SAXS and wide angle X-ray scattering experiments. The general performance of the 4C2 BL was initially tested using standard samples and further confirmed by the experience of users during three years of operation. In addition, several grazing incidence X-ray scattering measurements were carried out at the 4C2 BL.

pH-Dependent Structures of an i-Motif DNA in Solution

We have investigated for the first time the structure of i-motif DNA in solution at various pH conditions by using synchrotron small-angle X-ray scattering technique. To facilitate direct structural comparison between solution structures of i-motif DNA at various pH values, we created atomic coordinates of i-motif DNA from a fully folded to unfolded atomic model. Under mild acidic conditions, the conformations for i-motif DNA appeared to be similar to that of the partially unfolded i-motif atomic model in overall shape, rather than the fully folded i-motif atomic model. Collectively, our observations indicate that i-motif DNA molecule is structurally dynamic over a wide pH range, adopting multiple conformations ranging from the folded i-motif structure to a random coil conformation. As the i-motif structure has been used as an important component in nanomachines, we can therefore believe that the structural evidence presented herein will promote the development of future DNA-based molecular-actuator devices.

Crystal structure of RseB and a model of its binding mode to RseA

The bacterial envelope stress response senses stress signals in the extracytoplasmic compartment, and activates σE-dependent transcription by degrading its antisigma factor RseA. RseB, a binding partner of RseA, plays a pivotal role in regulating this response, but its molecular mechanism is not understood. We therefore determined the crystal structure of Escherichia coli RseB at a resolution of 2.4 Å. RseB is composed of two domains linked by a flexible linker and forms a loosely packed dimer with two grooves on each side. This structural feature is confirmed by small-angle scattering in solution. Analysis of the binding of various RseA mutants to RseB allowed us to identify the major RseB-binding motif in RseA. These data, coupled with analysis of small-angle scattering of the RseA/RseB complex in solution, leads us to propose that two RseAs bind to the grooves of the dimeric RseB by conserved residues. The implications for modulating proteolytic cleavage of RseA are discussed.

Structural evolution in microbial polyesters.

The crystallization behavior of microbially synthesized poly(3-hydroxybutyrate) (PHB) and its copolymers [P(HB-co-HHx)] containing 2.5, 3.4, and 12 mol % 3-hydroxyhexanoate (HHx) comonomer and the melting of the resultant crystals were studied in detail using time-resolved small-angle X-ray scattering and differential scanning calorimetry. The polyesters were found to undergo primary crystallization as well as secondary crystallization. In the primary crystallization, the thicknesses of the lamellar crystals were sensitive to the crystallization temperature, but no thickening was observed throughout the entire crystallization at a given temperature. The thickness of the lamellar crystals in the PHB homopolymer was always larger than that of the amorphous layers. In the copolymers, by contrast, the randomly distributed HHx comonomer units were found to be excluded from the lamellar crystals into the amorphous regions during the isothermal crystallization process. This interrupted the crystallization of the copolymer chains, resulting in the formation of lamellar crystals with thicknesses smaller than those of the amorphous layers. The lamellar crystals in the copolymers had lower electron densities compared to those formed in the PHB homopolymer. On the other hand, secondary crystallization favorably occurred during the later stage of isothermal crystallization in competition with the continuous primary crystallization, forming secondary crystals in amorphous regions, in particular in the amorphous layers between the primarily formed lamellar crystal stacks. Compared to the primarily formed lamellar crystals, the secondary crystals had short-range-ordered structures of smaller size, a broader size distribution, and a lower electron density.

Synchrotron Small-Angle X-ray Scattering Studies of the Structure of Porcine Pepsin under Various pH Conditions

Structural characteristics of various conformational states of porcine pepsin in solution under different pH conditions were investigated in terms of size and shape by small-angle X-ray scattering (SAXS). Low-resolution structural models of porcine pepsin were reconstructed from SAXS data, which were made inside the search volume of maximum dimension (Dmax), calculated from the pair distance distribution function p(r). The reconstructed structural models were obtained without imposing any restrictions on the symmetry or anisometry of the pepsin molecule. Under conditions emulating those for physiological activity of the enzyme, the reconstructed structural models exhibited a more extended C-terminal domain compared to the crystal structure. The differences between the solution and crystal structures of pepsin can be explained by inherent conformations of the flexible subdomain in the C-terminal domain under the solution pH conditions. Under mild acidic conditions where the enzyme is inactive, the reconstructed structural models revealed a compact globular conformation similar in overall shape to the crystal structure. These results indicate that the changes in fluorescence and circular dichroism curves observed under acidic conditions could also arise from the inherent conformation of the flexible subdomain, which has a tendency to roll into a sphere in the overall structure, but without affecting the stability of internal structure. Furthermore, the conformational changes in the subdomain might explain the inactivity of pepsin under mildly acidic conditions. Finally, compared to neutral denaturing conditions, pepsin under alkaline denaturing conditions had a larger expanded vertical conformation in the reconstructed model, as a consequence of alkaline denaturation of the N-terminal domain and a fully extended conformation of the C-terminal domain. The structural evidence presented here may have important implications for understanding the relationship between the structure of porcine pepsin and enzymatic function.

Two-Dimensionally Well-Ordered Multilayer Structures in Thin Films of a Brush Polypeptide

In this study, we report the first production of two-dimensionally well-ordered molecular multilayers (i.e., with a well-defined molecular lamellar structure) based on the antiparallel beta-sheet chain conformation in thin films of a brush polypeptide, poly(S-n-hexadecyl-dl-homocysteine) (PHHC), through the use of a simple spin-coating process and the quantitative structural and property analysis of the thin films using a grazing incidence X-ray scattering technique combined with Fourier transform infrared spectroscopy and differential scanning calorimetry. These analyses provide detailed information about the structure and molecular conformation of the self-assembled lamellae in the PHHC thin film, which is not easily obtained using conventional techniques. Moreover, we used the in situ measurements carried out at various temperatures and the data analyses to establish mechanisms for the evolution of the self-assembled lamellar structures in the film and for their melting. In addition, we propose molecular structure models of the PHHC polymer molecules in the thin film at various temperatures.

Probing conformational change of intrinsically disordered α-synuclein to helical structures by distinctive regional interactions with lipid membranes.

α-Synuclein (α-Syn) is an intrinsically disordered protein, whose fibrillar aggregates are associated with the pathogenesis of Parkinsons disease. α-Syn associates with lipid membranes and forms helical structures upon membrane binding. In this study, we explored the helix formation of α-Syn in solution containing trifluoroethanol using small-angle X-ray scattering and electrospray ionization ion mobility mass spectrometry. We then investigated the structural transitions of α-Syn to helical structures via association with large unilamellar vesicles as model lipid membrane systems. Hydrogen-deuterium exchange combined with electrospray ionization mass spectrometry was further utilized to understand the details of the regional interaction mechanisms of α-Syn with lipid vesicles based on the polarity of the lipid head groups. The characteristics of the helical structures were observed with α-Syn by adsorption onto the anionic phospholipid vesicles via electrostatic interactions between the N-terminal region of the protein and the anionic head groups of the lipids. α-Syn also associates with zwitterionic lipid vesicles and forms helical structures via hydrophobic interactions. These experimental observations provide an improved understanding of the distinct structural change mechanisms of α-Syn that originate from different regional interactions of the protein with lipid membranes and subsequently provide implications regarding diverse protein-membrane interactions related to their fibrillation kinetics.

Natural Form of Noncytolytic Flexible Human Fc as a Long-Acting Carrier of Agonistic Ligand, Erythropoietin

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained “Y-shaped” structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUClast). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.