Shaoning Yu

Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy

Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I–VII). Amide I bands (1,700–1,600 cm−1) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both H2O and deuterium oxide (D2O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg ml−1) are needed in this protocol; typically, the procedure can be completed in 1–2 d.

One-pot synthesis of water-dispersible Ag2S quantum dots with bright fluorescent emission in the second near-infrared window

The second near-infrared window (NIR-II, wavelength of 1.0-1.4 μm) is optimal for the bioimaging of live animals due to their low albedo and endogenous autofluorescence. Herein, we report a facile and one-pot biomimetic synthesis approach to prepare water-dispersible NIR-II-emitting ultrasmall Ag(2)S quantum dots (QDs). Photoluminescence spectra showed that the emission peaks could be tuned from 1294 to 1050 nm as the size of the Ag(2)S QDs varied from 6.8 to 1.6 nm. The x-ray diffraction patterns and x-ray photoelectron spectra confirmed that the products were monoclinic α-Ag(2)S. Fourier transform infrared spectrograph analysis indicated that the products were protein-conjugated Ag(2)S QDs. Examination of cytotoxicity and the hemolysis test showed that the obtained Ag(2)S QDs had good biocompatibility, indicating that such a nanomaterial could be a new kind of fluorescent label for in vivo imaging.

Structure of apo-CAP reveals that large conformational changes are necessary for DNA binding

The binding of cAMP to the Escherichia coli catabolite gene activator protein (CAP) produces a conformational change that enables it to bind specific DNA sequences and regulate transcription, which it cannot do in the absence of the nucleotide. The crystal structures of the unliganded CAP containing a D138L mutation and the unliganded WT CAP were determined at 2.3 and 3.6 Å resolution, respectively, and reveal that the two DNA binding domains have dimerized into one rigid body and their two DNA recognition helices become buried. The WT structure shows multiple orientations of this rigid body relative to the nucleotide binding domain supporting earlier biochemical data suggesting that the inactive form exists in an equilibrium among different conformations. Comparison of the structures of the liganded and unliganded CAP suggests that cAMP stabilizes the active DNA binding conformation of CAP through the interactions that the N6 of the adenosine makes with the C-helices. These interactions are associated with the reorientation and elongation of the C-helices that precludes the formation of the inactive structure.

MRI‐Visualized, Dual‐Targeting, Combined Tumor Therapy Using Magnetic Graphene‐Based Mesoporous Silica

Targeting peptide-modified magnetic graphene-based mesoporous silica (MGMSPI) are synthesized, characterized, and developed as a multifunctional theranostic platform. This system exhibits many merits, such as biocompatibility, high near-infrared photothermal heating, facile magnetic separation, large T2 relaxation rates (r2), and a high doxorubicin (DOX) loading capacity. In vitro and in vivo results demonstrate that DOX-loaded MGMSPI (MGMSPID) can integrate magnetic resonance imaging, dual-targeting recognition (magnetic targeting and receptor-mediated active targeting), and chemo-photothermal therapy into a single system for a visualized-synergistic therapy of glioma. In addition, it is observed that the MGMSPID system has heat-stimulated, pH-responsive, sustained release properties. All of these characteristics would provide a robust multifunctional theranostic platform for visualized glioma therapy.

The Secondary Structure of Calcineurin Regulatory Region and Conformational Change Induced by Calcium/Calmodulin Binding

The protein serine/threonine phosphatase calcineurin (CN) is activated by calmodulin (CaM) in response to intracellular calcium mobilization. A widely accepted model for CN activation involves displacement of the CN autoinhibitory peptide (CN467–486) from the active site upon binding of CaM. However, CN activation requires calcium binding both to the low affinity sites of CNB and to CaM, and previous studies did not dissect the individual contributions of CNB and CaM to displacement of the autoinhibitory peptide from the active site. In this work we have produced separate CN fragments corresponding to the CNA regulatory region (CNRR381–521, residues 381–521), the CNA catalytic domain truncated at residue 341, and the CNA-CNB heterodimer with CNA truncated at residue 380 immediately after the CNB binding helix. We show that the separately expressed regulatory region retains its ability to inhibit CN phosphatase activity of the truncated CN341 and CN380 and that the inhibition can be reversed by calcium/CaM binding. Tryptophan fluorescence quenching measurements further indicate that the isolated regulatory region inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The results provide new support for a model in which calcium binding to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. Moreover, the secondary structural content of the CNRR381–521 was tentatively addressed by Fourier transform infrared spectroscopy. The results indicate that the secondary structure of CNRR381–521 fragment is predominantly random coil, but with significant amount of β-strand and α-helix structures.

Calcium-induced changes in calmodulin structural dynamics and thermodynamics

The thermodynamics of the interaction between Ca(2+) and calmodulin (CaM) was examined using isothermal titration calorimetry (ITC). The chemical denaturation of calmodulin was monitored spectroscopically to determine the stability of Ca(2+)-free (apo) and Ca(2+)-loaded (holo) CaMs. We explored the conformational and structural dynamics of CaM using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared (FT-IR) spectroscopy. The results of H-D exchange and FT-IR suggest that CaM activation by Ca(2+) binding involves significant conformational changes. The results have also revealed that while the overall conformation of holo-CaM is more stable than that of the apo-CaM, some part of its α-helix structures, most likely the EF-hand domain region, has more solvent exposure, thus, has a faster H-D exchange rate than that of the apo-CaM. The ITC method provides a new strategy for obtaining site-specific Ca(2+) binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.

Ultrasmall gadolinium hydrated carbonate nanoparticle: an advanced T1 MRI contrast agent with large longitudinal relaxivity

Inorganic nanoparticle-based T1 contrast agents with high longitudinal relaxivity (r1) and low r2/r1 ratio have attracted great interest in recent years. However, the r1 relaxivity of inorganic nanoparticles reported to date is relatively low. In this work, 2.3 ± 0.1 nm paramagnetic gadolinium hydrated carbonate nanoparticles (GHC-1) with a high r1 relaxivity of 34.8 mM-1 s-1 and low r2/r1 ratio of 1.17 are synthesized using a one-pot hydrothermal process. The r1 of GHC-1 is 9.4 times higher than that of Gd-DTPA at 0.55 T. The synthetic procedure is simple, cost effective, and easy to scale up. The nanoparticles have a small core size, an amorphous phase, and are well-coated by poly(acrylic acid). Due to the hydrophilic polymer coating, the particles are highly dispersible and stable in aqueous solution. No significant cellular or in vivo toxicity are observed for the nanoparticles, which guarantees the in vivo application of this material. Finally, we apply the nanoparticles to in vivo magnetic resonance imaging and study the biodistribution in organs. This study reveals GHC-1 as a potential candidate for a T1 contrast agent with extraordinary ability to enhance MR images.

Overestimated accuracy of circular dichroism in determining protein secondary structure.

Circular dichroism (CD) is a spectroscopic technique widely used for estimating protein secondary structures in aqueous solution, but its accuracy has been doubted in recent work. In the present paper, the contents of nine globular proteins with known secondary structures were determined by CD spectroscopy and Fourier transform infrared spectroscopy (FTIR) in aqueous solution. A large deviation was found between the CD spectra and X-ray data, even when the experimental conditions were optimized. The content determined by FTIR was in good agreement with the X-ray crystallography data. Therefore, CD spectra are not recommended for directly calculating the content of a protein’s secondary structure.

Probing the Ca2+/CaM-induced secondary structural and conformational changes in calcineurin

Calcineurin (CN) is a Ca(2+)/CaM-dependent Ser/Thr protein phosphatase that plays a critical role in coupling Ca(2+) signals to a cellular response. Various methods have been applied to explore CN activation. A widely accepted model involves CaM binding to the CaM-binding domain (CN 389-413), inducing displacement of the CN autoinhibitory peptide (CN 467-486) from the active site. However, almost the entire regulatory region (CN 374-521), except the autoinhibitory peptide, is not visible in the electron density map of the reported structures. In the present study, we determined the overall secondary structure of CN in the presence or absence of Ca(2+)/CaM using FT-IR, and the Ca(2+)/CaM-induced structural dynamics and conformational changes were monitored by hydrogen-deuterium exchange experiments. The results revealed that the regulatory domain possessed some intrinsic structure. The binding of Ca(2+) and subsequent binding of CaM generated a sequential folding of CN, transforming it into a more constrained, less flexible conformation.

Calcium-dependent conformational transition of calmodulin determined by Fourier transform infrared spectroscopy

The Ca(2+)-induced conformational changes in calmodulin (CaM) were monitored by Fourier transform infrared spectroscopy (FT-IR) at different molar ratios of Ca(2+) to CaM. The results show that these changes occur in two distinctive transitions. The first transition involves significant changes in the overall secondary structure with a small gain in solvent accessibility, and is completed after the second Ca(2+) binds to both EF-hands of its C-terminal domain. The second transition is accompanied by CaM folding into a tighter, less hydrogen-exchangeable structure, and is completed by the addition of the fourth Ca(2+) to have four Ca(2+) per molecule. Particularly, α-helices in CaM-nCa(2+)(n=0, 1, 2) are less stable than those in CaM-nCa(2+)(n=3, 4).