The Ribosome Modulates Nascent Protein Folding Kaiser, Christian M.; Goldman, Daniel H.; Chodera, John D. ...
Science (American Association for the Advancement of Science),
12/2011, Letnik:
334, Številka:
6063
Journal Article
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Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been ...extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state.
Tethering proteins to force probes, typically micrometer-sized beads, is a prerequisite for dissecting their properties with optical tweezers. DNA handles serve as spacers between the tethered ...protein of interest and the bead surface. Attachment sites of the DNA handles to both the surface of beads and to the protein of interest must be mechanically stable for optical tweezers experiments. The most prominent method for attaching DNA handles to proteins utilizes thiol chemistry, linking modified DNA to engineered cysteines in the target protein. This method, although experimentally straightforward, is impractical for the large number of proteins that endogenously contain multiple or essential cysteines at undesired positions. Here, we describe two alternative approaches that take advantage of genetically encoded tag sequences in the target protein. The first method uses the enzymes Sfp and BirA, and the second uses the more recently described SpyTag-SpyCatcher system. We outline the process of generating the DNA handles themselves, as well as how to make the DNA-protein chimeras for carrying out optical tweezers experiments. These methods have robustly worked for several diverse and complex proteins, including ones that are difficult to produce or purify, and for protein-containing complexes such as the ribosome. They will be useful in cases where chemistry-based approaches are impractical or not feasible.
How plant roots cope with the soil complexity and integrate heterogeneous conditions into development, defence, and metabolism remains unclear. Structured microfluidic devices now enable controlled ...generation of complex microenvironments for microscopy-based root studies.
Abstract
When interacting with the environment, plant roots integrate sensory information over space and time in order to respond appropriately under non-uniform conditions. The complexity and dynamic properties of soil across spatial and temporal scales pose a significant technical challenge for research into the mechanisms that drive metabolism, growth, and development in roots, as well as on inter-organismal networks in the rhizosphere. Synthetic environments, combining microscopic access and manipulation capabilities with soil-like heterogeneity, are needed to elucidate the intriguing antagonism that characterizes subsurface ecosystems. Microdevices have provided opportunities for innovative approaches to observe, analyse, and manipulate plant roots and advanced our understanding of their development, physiology, and interactions with the environment. Initially conceived as perfusion platforms for root cultivation under hydroponic conditions, microdevice design has, in recent years, increasingly shifted to better reflect the complex growth conditions in soil. Heterogeneous micro-environments have been created through co-cultivation with microbes, laminar flow-based local stimulation, and physical obstacles and constraints. As such, structured microdevices provide an experimental entry point into the complex network behaviour of soil communities.
Driver inattention and distraction are the main causes of road accidents, many of which result in fatalities. To reduce road accidents, the development of information systems to detect driver ...inattention and distraction is essential. Currently, distraction detection systems for road vehicles are not yet widely available or are limited to specific causes of driver inattention such as driver fatigue. Despite the increasing automation of driving due to the availability of increasingly sophisticated assistance systems, the human driver will continue to play a longer role as supervisor of vehicle automation. With this in mind, we review the published scientific literature on driver distraction detection methods and integrate the identified approaches into a holistic framework that is the main contribution of the paper. Based on published scientific work, our driver distraction detection framework contains a structured summary of reviewed approaches for detecting the three main distraction detection approaches: manual distraction, visual distraction, and cognitive distraction. Our framework visualizes the whole detection information chain from used sensors, measured data, computed data, computed events, inferred behavior, and inferred distraction type. Besides providing a sound summary for researchers interested in distracted driving, we discuss several practical implications for the development of driver distraction detection systems that can also combine different approaches for higher detection quality. We think our research can be useful despite - or even because of - the great developments in automated driving.
Abstract
Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of ...proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.
Driver behaviour monitoring is a broad area of research, with a variety of methods and approaches. Distraction from the use of electronic devices, such as smartphones for texting or talking on the ...phone, is one of the leading causes of vehicle accidents. With the increasing number of sensors available in vehicles, there is an abundance of data available to monitor driver behaviour, but it has only been available to vehicle manufacturers and, to a limited extent, through proprietary solutions. Recently, research and practice have shifted the paradigm to the use of smartphones for driver monitoring and have fuelled efforts to support driving safety. This systematic review paper extends a preliminary, previously carried out author-centric literature review on smartphone-based driver monitoring approaches using snowballing search methods to illustrate the opportunities in using smartphones for driver distraction detection. Specifically, the paper reviews smartphone-based approaches to distracted driving behaviour detection, the smartphone sensors and detection methods applied, and the results obtained.
The coupling of protein synthesis and folding is a crucial yet poorly understood aspect of cellular protein folding. Over the past few years, it has become possible to experimentally follow and ...define protein folding on the ribosome, revealing principles that shape co‐translational folding and distinguish it from refolding in solution. Here, we highlight some of these recent findings from biochemical and biophysical studies and their potential significance for cellular protein biogenesis. In particular, we focus on nascent chain interactions with the ribosome, interactions within the nascent protein, modulation of translation elongation rates, and the role of mechanical force that accompanies nascent protein folding. The ability to obtain mechanistic insight in molecular detail has set the stage for exploring the intricate process of nascent protein folding. We believe that the aspects discussed here will be generally important for understanding how protein synthesis and folding are coupled and regulated.
Many proteins begin to fold during translation. This sequential co‐translational folding is important to avoid protein aggregation and misfolding. Generation of mechanical force by nascent chain folding and denaturation of co‐translationally formed structures are among aspects of co‐translational folding that have recently come to light.
Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A ...substantial fraction of all cellular proteomes is composed of multidomain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multidomain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full‐length protein. The N‐terminal G‐domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non‐native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non‐native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multidomain proteins.
AAA
+ unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from
E. coli, ...alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.
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► ClpX generates force, most likely unfolding substrates as a power-stroke motor ► ClpX subunits take 1 nm steps and display a limited degree of coordination ► ClpX stochastically slips, briefly disengaging from its substrates ► ClpXP exhibits reduced substrate slippage and more robust protein unfolding than ClpX
Colorectal adenoma are precursor lesions on the pathway to cancer. Their removal in screening colonoscopies has markedly reduced rates of cancer incidence and death. Generic models of adenoma growth ...and transition to cancer can guide the implementation of screening strategies. But adenoma shape has rarely featured as a relevant risk factor. Against this backdrop we aim to demonstrate that shape influences growth dynamics and cancer risk. Stochastic cell-based models are applied to a data set of 197,347 Bavarian outpatients who had colonoscopies from 2006-2009, 50,649 patients were reported with adenoma and 296 patients had cancer. For multi-stage clonal expansion (MSCE) models with up to three initiating stages parameters were estimated by fits to data sets of all shapes combined, and of sessile (70% of all adenoma), peduncular (17%) and flat (13%) adenoma separately for both sexes. Pertinent features of adenoma growth present themselves in contrast to previous assumptions. Stem cells with initial molecular changes residing in early adenoma predominantly multiply within two-dimensional structures such as crypts. For these cells mutation and division rates decrease with age. The absolute number of initiated cells in an adenoma of size 1 cm is small around 103, related to all bulk cells they constitute a share of about 10-5. The notion of very few proliferating stem cells with age-decreasing division rates is supported by cell marker experiments. The probability for adenoma transiting to cancer increases with squared linear size and shows a shape dependence. Compared to peduncular and flat adenoma, it is twice as high for sessile adenoma of the same size. We present a simple mathematical expression for the hazard ratio of interval cancers which provides a mechanistic understanding of this important quality indicator. We conclude that adenoma shape deserves closer consideration in screening strategies and as risk factor for transition to cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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