Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding

Protein engineering was used previously to convert maltose-binding protein (MBP) into a zinc biosensor. Zn(2+) binding by the engineered MBP was thought to require a large conformational change from "open" to "closed", similar to that observed when maltose is bound by the wild-type protein. We show that although this re-designed MBP molecule binds Zn(2+) with high affinity as previously reported, it does not adopt a closed conformation in solution as assessed by small-angle X-ray scattering. High-resolution crystallographic studies of the engineered Zn(2+)-binding MBP molecule demonstrate that Zn(2+) is coordinated by residues on the N-terminal lobe only, and therefore Zn(2+) binding does not require the protein to adopt a fully closed conformation. Additional crystallographic studies indicate that this unexpected Zn(2+) binding site can also coordinate Cu(2+) and Ni(2+) with only subtle changes in the overall conformation of the protein. This work illustrates that the energetic barrier to domain closure, which normally functions to maintain MBP in an open concentration in the absence of ligand, is not easily overcome by protein design. A comparison to the mechanism of maltose-induced domain rearrangement is discussed.     

The cooperative voltage sensor motion that gates a potassium channel

The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.     

Rapid and accurate structure determination of coiled-coil domains using NMR dipolar couplings: application to cGMP-dependent protein kinase Ialpha

Coiled-coil motifs play essential roles in protein assembly and molecular recognition, and are therefore the targets of many ongoing structural and functional studies. However, owing to the dynamic nature of many of the smaller coiled-coil domains, crystallization for X-ray studies is very challenging. Determination of elongated structures using standard NMR approaches is inefficient and usually yields low-resolution structures due to accumulation of small errors over long distances. Here we describe a solution NMR approach based on residual dipolar couplings (RDCs) for rapid and accurate structure determination of coiled-coil dimers. Using this approach, we were able to determine the high-resolution structure of the coiled-coil domain of cGMP-dependent protein kinase Ialpha, a protein of previously unknown structure that is critical for physiological relaxation of vascular smooth muscle. This approach can be extended to solve coiled-coil structures with higher order assemblies.     

NMR characterization of the Escherichia coli nitrogen regulatory protein IIANtr in solution and interaction with its partner protein, NPr

The solution form of IIA(Ntr) from Escherichia coli and its interaction with its partner protein, NPr, were characterized by nuclear magnetic resonance (NMR) spectroscopy. The diffusion coefficient of the protein (1.13 x 10(-6) cm/sec) falls between that of HPr (approximately 9 kDa) and the N-terminal domain of E. coli enzyme I (approximately 30 kDa), indicating that the functional form of IIA(Ntr) is a monomer (approximately 18 kDa) in solution. Thus, the dimeric structure of the protein found in the crystal is an artifact of crystal packing. The residual dipolar coupling data of IIA(Ntr) (covering residues 11-155) measured in the absence and presence of a 4% polyethyleneglycol-hexanol liquid crystal alignment medium fit well to the coordinates of both molecule A and molecule B of the dimeric crystal structure, indicating that the 3D structures in solution and in the crystal are indeed similar for that protein region. However, only molecule A possesses an N-terminal helix identical to that derived from chemical shifts of IIA(Ntr) in solution. Further, the (15)N heteronuclear nuclear Overhauser effect (NOE) data also support molecule A as the representative structure in solution, with the terminal residues 1-8 and 158-163 more mobile. Chemical shift mapping identified the surface on IIA(Ntr) for NPr binding. Residues Gly61, Asp115, Ser125, Thr156, and nearby regions of IIA(Ntr) are more perturbed and participate in interaction with NPr. The active-site His73 of IIA(Ntr) for phosphoryl transfer was found in the Ndelta1-H tautomeric state. This work lays the foundation for future structure and function studies of the signal transducing proteins from this nitrogen pathway.     

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.     

Solid-phase extraction or liquid chromatography coupled on-line with gas chromatography in the analysis of biological samples

This review provides an overview of the on-line coupling of solid-phase extraction or liquid chromatography with gas chromatography for the analysis of biological samples. Principles relevant to techniques are briefly presented and selected applications are described. Benefits of the coupled systems are discussed.     

Human impacts decouple a fundamental ecological relationship—The positive association between host diversity and parasite diversity

Human impacts on ecosystems can decouple the fundamental ecological relationships that create patterns of diversity in free‐living species. Despite the abundance, ubiquity, and ecological importance of parasites, it is unknown whether the same decoupling effects occur for parasitic species. We investigated the influence of fishing on the relationship between host diversity and parasite diversity for parasites of coral reef fishes on three fished and three unfished islands in the central equatorial Pacific. Fishing was associated with a shallowing of the positive host‐diversity–parasite‐diversity relationship. This occurred primarily through negative impacts of fishing on the presence of complex life‐cycle parasites, which created a biologically impoverished parasite fauna of directly transmitted parasites resilient to changes in host biodiversity. Parasite diversity appears to be decoupled from host diversity by fishing impacts in this coral reef ecosystem, which suggests that such decoupling might also occur for parasites in other ecosystems affected by environmental change.     

患者来源的异种移植模型：促进前列腺癌转化研究的新平台

近年来,患者来源异种移植模型(Patient-derived xenograft,PDX)引起了科研工作者的广泛关注。PDX模型在生物学的研究、临床前药物开发及个性化治疗策略的制定等方面比传统的细胞移植肿瘤模型有着更显著的意义,其特点在于保留了人来源肿瘤组织的分子生物学、基因及组织学特性。目前,国外多个实验室已经建立了多种肿瘤PDX模型,本文综述了当前这一领域的研究进展,并对PDX模型在前列腺癌研究中的优势及尚需解决的问题进行了阐述。     

Thermodynamic secrets of multidrug resistance: A new take on transport mechanisms of secondary active antiporters

Multidrug resistance (MDR) presents a growing challenge to global public health. Drug extrusion transporters play a critical part in MDR; thus, their mechanisms of substrate recognition are being studied in great detail. In this work, we review common structural features of key transporters involved in MDR. Based on our membrane potential‐driving hypothesis, we propose a general energy‐coupling mechanism for secondary‐active antiporters. This putative mechanism provides a common framework for understanding poly‐specificity of most—if not all—MDR transporters.     

Safety and Efficacy of Apatinib Combined with Temozolomide in Advanced Melanoma Patients after Conventional Treatment Failure

OBJECTIVE: Asian melanoma patients, predominantly comprised of acral and mucosal subtypes, might not benefit from immunotherapy and targeted therapy as much as Caucasian patients. Novel treatment strategies are demanded after conventional treatment failure. This was a prospective, single-arm, and single-center dose escalation study to investigate the safety and preliminary efficacy of apatinib combined with temozolomide in heavily treated advanced melanoma patients. METHODS: Patients were sequentially admitted to four dose-escalating groups of apatinib and temozolomide (three cases in each group) using a traditional 3?+?3 dose escalation design method. RESULTS: Twelve patients were enrolled between December 2016 and August 2017. Most patients with an acral or mucosal primary origin progressed after immunotherapy or targeted therapy. Dose escalation had been completed without dose-limiting toxicity. Common adverse events included hypertension, hand-foot syndrome, proteinuria, neutropenia, nausea, and fatigue. All adverse events were grade 1 or 2, while the maximum tolerated dose was not reached. Up to January 2018, 1 patient achieved partial response, 9 experienced stable disease, and 2 exhibited progressive disease. The objective response rate and disease control rate were 8.3% and 83%, respectively. CONCLUSIONS: In conclusion, apatinib combined with temozolomide was well tolerated and has demonstrated efficacy in advanced melanoma patients.