Effect of crowding by Ficolls on OmpA and OmpT refolding and membrane insertion

Folding of outer membrane proteins (OMPs) has been studied extensively in vitro. However, most of these studies have been conducted in dilute buffer solution, which is different from the crowded environment in the cell periplasm, where the folding and membrane insertion of OMPs actually occur. Using OmpA and OmpT as model proteins and Ficoll 70 as the crowding agent, here we investigated the effect of the macromolecular crowding condition on OMP membrane insertion. We found that the presence of Ficoll 70 significantly slowed down the rate of membrane insertion of OmpA while had little effect on those of OmpT. To investigate if the soluble domain of OmpA slowed down membrane insertion in the presence of the crowding agent, we created a truncated OmpA construct that contains only the transmembrane domain (OmpA171). In the absence of crowding agent, OmpA171 refolded at a similar rate as OmpA, although with decreased efficiency. However, under the crowding condition, OmpA171 refolded significantly faster than OmpA. Our results suggest that the periplasmic domain slows down the rate, while improves the efficiency, of OmpA folding and membrane insertion under the crowding condition. Such an effect was not obvious when refolding was studied in buffer solution in the absence of crowding.     

Amide proton exchange of a dynamic loop in cell extracts

Intrinsic rates of exchange are essential parameters for obtaining protein stabilities from amide ¹H exchange data. To understand the influence of the intracellular environment on stability, one must know the effect of the cytoplasm on these rates. We probed exchange rates in buffer and in Escherichia coli lysates for the dynamic loop in the small globular protein chymotrypsin inhibitor 2 using a modified form of the nuclear magnetic resonance experiment, SOLEXSY. No significant changes were observed, even in 100 g dry weight L⁻¹ lysate. Our results suggest that intrinsic rates from studies conducted in buffers are applicable to studies conducted under cellular conditions.     

Current developments in Coot for macromolecular model building of Electron Cryo‐microscopy and Crystallographic Data

Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo‐EM model building and refinement, as cryo‐EM structures with resolution ranging 2.5–4 A are now routinely available. Model building into these maps can be time‐consuming and requires experience in both biochemistry and building into low‐resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman‐McClure restraints, full‐chain refinement, and Fourier‐model based residue‐type‐specific Ramachandran restraints. Here, we present the current state‐of‐the‐art in Coot usage.     

Why is the microtubule lattice helical?

Microtubules polymerize from identical tubulin heterodimers, which form a helical lattice pattern that is the microtubule. This pattern always has left-handed chirality, but it is not known why. But as tubulin, similar to other proteins, evolved for a purpose, the question of the title of this artcile appears to be meaningful. In a computer simulation that explores the 'counterfactual biology' of microtubules without helicity, we demonstrate that these have the same mechanical properties as Nature's microtubules with helicity. Thus only a dynamical reason for helicity is left as potential explanation. We find that helicity solves 'the problem of the blind mason', i.e. how to correctly build a structure, guided only by the shape of the bricks. This answer in turn raises some new questions for researchers to address.     

Thermodynamic theory explains the temperature optima of soil microbial processes and high Q10 values at low temperatures

Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory – MMRT (Hobbs et al., 2013 , ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q₁₀ calculations) at low temperatures and that Q₁₀ declined as temperature increases in agreement with data synthesis from the literature. Declining Q₁₀ and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme‐catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems – a fundamental component of models for climate change.     

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group^1-3. JoVE video articles describing the method are available^1,4. The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group^5,6. The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.     

Ultrafast electron transfer in riboflavin binding protein in macromolecular crowding of nano-sized micelle

In this contribution, we have studied the dynamics of electron transfer (ET) of a flavoprotein to the bound cofactor, an important metabolic process, in a model molecular/macromolecular crowding environments. Vitamin B₂ (riboflavin, Rf) and riboflavin binding protein (RBP) are used as model cofactor and flavoprotein, respectively. An anionic surfactant sodium dodecyl sulfate (SDS) is considered to be model crowding agent. A systematic study on the ET dynamics in various SDS concentration, ranging from below critical micellar concentration (CMC), where the surfactants remain as monomeric form to above CMC, where the surfactants self-assemble to form nanoscopic micelle, explores the dynamics of ET in the model molecular and macromolecular crowding environments. With energy selective excitation in picosecond-resolved studies, we have followed temporal quenching of the tryptophan residue of the protein and Rf in the RBP–Rf complex in various degrees of molecular/macromolecular crowding. The structural integrity of the protein (secondary and tertiary structures) and the vitamin binding capacity of RBP have been investigated using various techniques including UV–Vis, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) studies in the crowding environments. Our finding suggests that the effect of molecular/macromolecular crowding could have major implication in the intra-protein ET dynamics in cellular environments.     

Measuring how two proteins affect each other's net charge in a crowded environment

Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein''s charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester‐Staudinger pairs) can be used to mimic crowding by linking two non‐interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine‐acyl “protein charge ladders” and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z _monomer = −0.43 ± 0.01) and α‐lactalbumin (Z _monomer = −4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = −0.04 ± 0.09 upon crowding by this pair (Z _dimer = −5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.     

The effects of macromolecular crowding on the mechanical stability of protein molecules

Macromolecular crowding, a common phenomenon in the cellular environments, can significantly affect the thermodynamic and kinetic properties of proteins. A single-molecule method based on atomic force microscopy (AFM) was used to investigate the effects of macromolecular crowding on the forces required to unfold individual protein molecules. It was found that the mechanical stability of ubiquitin molecules was enhanced by macromolecular crowding from added dextran molecules. The average unfolding force increased from 210 pN in the absence of dextran to 234 pN in the presence of 300 g/L dextran at a pulling speed of 0.25 microm/sec. A theoretical model, accounting for the effects of macromolecular crowding on the native and transition states of the protein molecule by applying the scaled-particle theory, was used to quantitatively explain the crowding-induced increase in the unfolding force. The experimental results and interpretation presented could have wide implications for the many proteins that experience mechanical stresses and perform mechanical functions in the crowded environment of the cell.     

杨树和冬小麦在短日照和寒冬中的胞间连丝动态

电镜观察揭示,三角杨(Populus deltoides Marsh.)和冬小麦(Triticum aestivum L. cv.Seward 80004)的胞间连丝(PD)结构在8 h短日照和寒冷冬季里发生明显的变化,并在这两种植物之间表现出明显的区别.在16 h长日照条件下,杨树顶芽分生组织和冬小麦幼叶组织细胞壁中形成大量的PD,染色较深,并可看到细胞质中的内质网(ER)与PD中ER的连接;在冬小麦幼叶组织中还可观察到典型的"颈型"PD,其中央桥管(压扁的ER)清晰可见.在短日照条件下,杨树顶芽细胞壁中出现许多不完整的、中断的PD,有些PD在壁的中部发生膨胀,形成空腔,看不到中央桥管.然而,冬小麦幼叶组织中的PD不发生明显的改变,与长日照条件下的结构基本相似.在寒冷冬季里,杨树PD结构的变化与短日照的相似.冬小麦的PD也发生明显的变化,主要表现在两个方面:一是PD口被一种电子稠密物质堵塞;二是颈区口径缩小.PD的这些变化以及在这两种植物(木本和草本)之间的区别,证实了木本杨树和草本冬小麦对短日照反应和休眠状态的不同.短日照能诱发木本杨树基因表达的改变,产生结构和生理生化过程的变化,进入休眠,并在秋季低温共同作用下,进一步发展抗寒力.这里观察到杨树PD在短日照和寒冬中的中断正是从结构上导致其植株进入休眠和提高抗寒力的一个过程.冬小麦对短日照不起反应,低温则能诱发其抗寒基因表达,发展抗寒力,但不产生"生理休眠",所以它的PD在短日照下不发生变化,在低温下的改变则很容易恢复,当冬小麦在大气温度升高时,颈口的堵塞物质会迅速消失,口径也会迅速扩大,植株随即迅速恢复生长.