Structural and mechanistic consequences of polypeptide binding by GroEL

The remarkable ability of the chaperonin GroEL to recognise a diverse range of non-native states of proteins constitutes one of the most fascinating molecular recognition events in protein chemistry. Recent structural studies have revealed a possible model for substrate binding by GroEL and a high-resolution image of the GroEL–GroES folding machinery has provided important new insights into our understanding of the mechanism of action of this chaperonin. Studies with a variety of model substrates reveal that the binding of substrate proteins to GroEL is not just a passive event, but can result in significant changes in the structure and stability of the bound polypeptide. The potential impact of this on the mechanism of chaperonin-assisted folding is not fully understood, but provides exciting scope for further experiment

Characterization of the Response of Primary Cells Relevant to Dialysis-Related Amyloidosis to β2-Microglobulin Monomer and Fibrils

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of β2-microglobulin (β2m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar β2m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate β2m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade β2m fibrils, and that both monomeric β2m and fibrillar β2m are cytotoxic to these cells. β2m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that β2m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that β2m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that β2m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA

Unfolding dynamics of proteins under applied force

Understanding the mechanisms of protein folding is a major challenge that is being addressed effectively by collaboration between researchers in the physical and life sciences. Recently, it has become possible to mechanically unfold proteins by pulling on their two termini using local force probes such as the atomic force microscope. Here, we present data from experiments in which synthetic protein polymers designed to mimic naturally occurring polyproteins have been mechanically unfolded. For many years protein folding dynamics have been studied using chemical denaturation, and we therefore firstly discuss our mechanical unfolding data in the context of such experiments and show that the two unfolding mechanisms are not the same, at least for the proteins studied here. We also report unexpected observations that indicate a history effect in the observed unfolding forces of polymeric proteins and explain this in terms of the changing number of domains remaining to unfold and the increasing compliance of the lengthening unstructured polypeptide chain produced each time a domain unfolds

Protein folding in the cell: challenges and progress.

It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, cotranslational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, such as fluorescence imaging of folding in cells and genetic screens for in-cell stability. DOI 10.1016DOI 10. /j.sbi.2010 Introduction Chris Anfinsen launched the field of protein folding by showing that ribonuclease (specifically, bovine pancreatic ribonuclease A) could refold to an active enzyme after reductive denaturation. Naturally, ribonuclease became emblematic of the fundamental tenet of protein folding -that the primary sequence of a protein specifies an energy landscape and a successful route to the native state at the global energy minimum. Yet ribonuclease folds in vivo during a complex journey through the secretory pathway of the cell. Notably, in its biological folding process, ribonuclease confronts milieux that are densely crowded with macromolecules; it samples the microenvironments of the ribosome tunnel, the translocon, and the ER lumen; it has the opportunity to fold from its N-terminus to C-terminus; and it is not left on its own, but instead is accompanied by lumenal chaperones that facilitate its folding and post-translational modification. As this journey abundantly illustrates, protein folding in the cell confronts many issues that are nonexistent in high dilution refolding experiments. It is thus not surprising that an increasing research effort is being applied to issues and processes involved in cellular protein folding. This review presents research in the expanding area of protein folding in the cell. We mainly confine our discussion to publications on cellular protein folding that have appeared in the last two years and to areas that have not been recently reviewed. We first describe a number of issues, such as macromolecular crowding and cotranslational folding, that arise when considering folding in the cell but are absent in vitro ( Macromolecular crowding A striking difference between most in vitro folding experiments and the cellular environment is the high concentration of macromolecules, which severely limits the cellular volume accessible to a polypeptide chain. Like many issues related to folding in the cell, determining the effects of crowding on folding presents major technical challenges to both computational and experimental studies; moreover, crowding is generally accompanied by other effects including altered diffusion and weak interactions. Macromolecular crowding also affects the viscosity of the cellular environment and solvent viscosity has been invoked as an important factor in determining folding rates and mechanisms (e.g., [9]). In a recent study, Dhar et al. used computational and experimental approaches to study the effects of a model crowder, Ficoll, on activity and folding of phosphoglycerate kinase (PGK), a 412-aa protein with two domains connected by a flexible hinge www.sciencedirect.com Current Opinion in Structural Biology 2011, 21:32-41 decreased the inter-domain separation. Also, the relaxation rate from temperature-jump unfolding experiments showed a maximum at 100 g/L Ficoll. The authors interpreted this result as arising from opposing effects of crowding and viscosity Hindered mobility and sticky neighbors Several recent studies show that the cellular environment affects macromolecular motion and provide insight into how. For example, two recent papers have used fluorescence recovery after photobleaching to monitor translational diffusion of GFP constructs in E. coli cells Vectorial synthesis and roles of mRNA and ribosomes in folding Newly synthesized polypeptide chains emerge from the ribosome vectorially, allowing their N-terminal portions to sample conformational space before the chain is completely synthesized. Additionally, the earliest environments encountered by a nascent chain are the ribosome tunnel and ribosome-associated chaperones. There have been excellent recent reviews on issues related to cotranslational folding including one in this issue A single domain stabilized by many long-range contacts is not expected to fold until the entire chain is complete, and recent studies of ribosome-bound nascent chains (RNCs) have confirmed this expectation for an SH3 domain by NMR [20] and GFP by observing chromophore maturation [21 ]. In fact, the NMR studies on SH3 RNCs reveal little or no compaction until the entire chain has exited the ribosomal tunnel [20]. By comparison, RNCs of the larger GFP may populate a more compact state before full translation [21 ]. The interrelationship of translation rate and folding has been discussed in a number of recent reviews [22][23][24][25]. Intuitively, slowing translation might allow more time for proper folding, and indeed in a recent study mutant ribosomes with reduced translation rates increased the soluble expression of eukaryotic proteins in E. coli [26 ]. The messenger RNA sequence can also affect the translation rate either through the use of rare codons [22,25,[27][28][29] or by RNA folding [29,30]. Either of these factors may be changed by synonymous mutations, where mRNA sequences are altered without affecting the encoded amino acid. Increasing the translation rate of the multidomain E. coli protein SufI by synonymously exchanging rare codon clusters for common codons was found to decrease cotranslational folding and the production of mature, folded protein [31 ]. Intriguingly, the mutation that leads to the deletion of F508 (DF508) in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common mutation linked to cystic fibrosis, also changes the preceding codon for Ile507 [32 ]. Alteration of the local mRNA structure in the mutant retards translation and presumably impairs folding, increasing cotranslational ubiquitination and leading to protein degradation [32 ,33]. Restoration of the original Ile507 codon in the DF508 background significantly increases the amount of mature CFTR in the plasma membrane, demonstrating the potential impact of synonymous mutations on in vivo protein folding and maturation [32 ,33]. As described in recent reviews Molecular chaperones remodel the in vivo folding energy landscape The ability of molecular chaperones to interact with nascent or incompletely folded chains so as to favor successful folding and disfavor aggregation is well established. Yet the impact of chaperones on the folding mechanisms and stabilities of their clients is less clear, despite expanding literature on the functions and substrate-omes of several chaperones (e.g., Consider the case of arguably the best-studied chaperone, the E. coli chaperonin GroEL and its partner GroES. On Spatial organization, membranes and compartmentalization Cellular interiors are highly anisotropic with elaborate and physiologically critical architectures. This subcellular organization plays a major role in folding at several levels. For example, the native structures of membrane proteins are tuned to the diverse microenvironments and twodimensional character of membranes. Additionally, membranes are barriers, requiring proteins made on one side but destined to perform their functions extracytoplasmically to be translocated across the membrane, in most cases unfolded. For secretory proteins, passage through cellular compartments is highly choreographed with an assembly line of modifying enzymes, chaperones and transport mechanisms. Compartmentalization also opens up the possibility of chemical gradients, for example pH or oxidizing potential. Taken together, the spatial organization and compartmentalization of the cellular environment enable folding to occur in temporally optimized steps. Bacterial proteins destined for the periplasm or the cell exterior can be secreted across membranes either folded (e.g., by the Tat system) or unfolded (e.g., by the Sec system) Proteins that translocate through the Sec channel in bacteria and eukaryotes are greeted by an array of chaperones and modifying enzymes that alter the folding energy landscape. In addition, they move from an ATPrich, reducing environment to one that is ATP-poor and oxidizing as they enter either the bacterial periplasm, mitochondrial intermembrane space (IMS) or the eukaryotic ER. We refer interested readers to recent reviews of protein folding in the ER Exposure of nascent chains to the oxidizing environment of the periplasm or ER lumen enables step-wise disulfide bond formation, fixing the topology of secretory proteins. Not surprisingly, the timing and specificity of disulfide bond formation is integral to their in vivo folding, and this issue has been widely studied in eukaryotic proteins Many proteins that fold in the ER lumen are large with complicated topologies including multiple domains, disulfides, and glyosylation sites, which play roles in specialized in vivo folding mechanisms. A recent study on the influenza membrane glycoprotein neuraminidase Promising new methods for the study of in vivo protein folding The Holy Grail in studies of protein folding in the cell is to directly observe a protein of interest (POI) in intact cells and to characterize its folding, both thermodynamically and kinetically, in situ. Not surprisingly, this has proven exceedingly difficult. Several years ago, Ghaemmaghami and Oas took advantage of E. coli's urea tolerance to perform in-cell urea titrations of the l repressor headpiece utilizing a novel hydrogen exchange/ mass spectrometry method to assess stability Exciting recent work from the Gruebele lab combines temperature-jump perturbation methods with fast relaxation imaging (FReI) to interrogate the in vivo folding landscape of a POI, here a temperature-sensitive variant of phosphoglycerate kinase (tsPGK) In-cell NMR, recently reviewed by Pielak et al. [70] and by Ito and Selenko [71], is a potentially powerful approach to study proteins in vivo and to gain insight into their stability and folding mechanisms. Unfortunately, many folded proteins fail to show measurable NMR spectra in the cellular environment, most likely because of hindered rotational diffusion. Despite the inherent obstacles, the Shirakawa group has had impressive success applying NMR to small proteins in eukaryotic cells Clever use of split reporters, in which folding of the POI is coupled to successful binding and folding of two pieces of 36 Folding and Binding Figure 2 Monitoring protein folding kinetics in a living cell using fast relaxation imaging (FReI) These approaches to in-cell stability use a single flanking reporter and are clearly powerful as selections for enhanced stability. However, they are end-point assays based on the proteolytic lability of the fusion construct when the POI is unstable and therefore cannot readily yield an estimate of folding free energy. A related in vivo screen flanks the POI between a DNA-binding domain and a transcriptional activation domai

Molecular Origins of the Compatibility Between Glycosaminoglycans and Aβ40 Amyloid Fibrils

The Aβ peptide forms extracellular plaques associated with Alzheimer's disease. In addition to protein fibrils, amyloid plaques also contain non-proteinaceous components, including glycosaminoglycans (GAGs). We have shown previously that the GAG low molecular weight heparin (LMWH) binds to Aβ40 fibrils with a three-fold-symmetric (3Q) morphology with higher affinity than Aβ40 fibrils in alternative structures, Aβ42 fibrils, or amyloid fibrils formed from other sequences. Solid-state NMR (SSNMR) analysis of the GAG-3Q fibril complex revealed an interaction site at the corners of the 3Q fibril structure, but the origin of the binding specificity remained obscure. Here, employing a library of short heparin polysaccharides modified at specific sites, we show that the NS or 6-OS glucosamine sulfates, but not the 2-OS iduronate sulfate, of heparin is required for 3Q binding, indicating selectivity in the interactions of the GAG with the fibril that extends beyond general electrostatic complementarity. By creating 3Q fibrils containing point substitutions in the amino acid sequence, we also show that charged residues at the fibril three-fold apices provide the majority of the binding free energy, while charged residues elsewhere are less critical for binding. The results indicate, therefore, that LMWH binding to 3Q fibrils requires a precise molecular complementarity of the sulfate moieties on the GAG and charged residues displayed on the fibril surface. Differences in GAG binding to fibrils with distinct sequence and/or structure may thus contribute to the diverse etiology and progression of amyloid diseases

Investigating the structural compaction of biomolecules upon transition to the gas-phase using ESI-TWIMS-MS

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein’s size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds

Data for "Mechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer Electrolyte Nanopore"

The data set contains all relevant ionic current traces and voltammetric data recorded using pClamp system for the associated paper entitled: 'Mechanistic Study of the Conductance and Enhanced Single-Molecule Detection in a Polymer Electrolyte Nanopore'. The data is in the native pClamp format of abf files. Analysis routine can be performed with script that relies on abf files. The data set is aim to allow users to re-analysis the data and to reproduce the observations made in the associated publication