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Advances in Protein Chemistry and... 2022The outer membrane of Gram-negative bacteria is a specialized organelle conferring protection to the cell against various environmental stresses and resistance to many... (Review)
Review
The outer membrane of Gram-negative bacteria is a specialized organelle conferring protection to the cell against various environmental stresses and resistance to many harmful compounds. The outer membrane has a number of unique features, including an asymmetric lipid bilayer, the presence of lipopolysaccharides and an individual proteome. The vast majority of the integral transmembrane proteins in the outer membrane belongs to the family of β-barrel proteins. These evolutionarily related proteins share a cylindrical, anti-parallel β-sheet core fold spanning the outer membrane. The loops and accessory domains attached to the β-barrel allow for a remarkable versatility in function for these proteins, ranging from diffusion pores and transporters to enzymes and adhesins. We summarize the current knowledge on β-barrel structure and folding and give an overview of their functions, evolution, and potential as drug targets.
Topics: Bacteria; Bacterial Outer Membrane Proteins; Lipid Bilayers; Protein Folding
PubMed: 35034717
DOI: 10.1016/bs.apcsb.2021.07.002 -
The International Journal of... Nov 2017The key role of monoamine transporters is to take up neurotransmitters from the synaptic cleft and rapidly terminate neurotransmission. Monoamine transporters begin... (Review)
Review
The key role of monoamine transporters is to take up neurotransmitters from the synaptic cleft and rapidly terminate neurotransmission. Monoamine transporters begin their journey by folding in the endoplasmic reticulum. Upon achieving their natively-folded state, the oligomerized transporters engage the coat protein complex II machinery and exit the endoplasmic reticulum compartment in a concentrative fashion. The transporters are subsequently sorted in the endoplasmic reticulum-Golgi intermediate complex and the Golgi apparatus, prior to reaching their pivotal site of action at the plasma membrane. Stringent quality-control mechanisms ensure that only the correctly-folded protein cargo departs the endoplasmic reticulum. Genetic point mutations in the coding sequences of monoamine transporters can trigger severe physiologic deficiencies by inducing folding defects. Protein misfolding precludes the delivery of functional monoamine transporters to the cell surface. Chemical- and/or pharmacological-chaperone molecules, which facilitate folding, have proven effective in restoring the activity of several misfolded pathological variants of monoamine transporters.
Topics: Animals; Endoplasmic Reticulum; Humans; Molecular Targeted Therapy; Neurotransmitter Transport Proteins; Protein Folding; Small Molecule Libraries
PubMed: 28890376
DOI: 10.1016/j.biocel.2017.09.004 -
BMC Bioinformatics Nov 2023Recently, significant progress has been made in the field of protein structure prediction by the application of artificial intelligence techniques, as shown by the...
BACKGROUND
Recently, significant progress has been made in the field of protein structure prediction by the application of artificial intelligence techniques, as shown by the results of the CASP13 and CASP14 (Critical Assessment of Structure Prediction) competition. However, the question of the mechanism behind the protein folding process itself remains unanswered. Correctly predicting the structure also does not solve the problem of, for example, amyloid proteins, where a polypeptide chain with an unaltered sequence adopts a different 3D structure.
RESULTS
This work was an attempt at explaining the structural variation by considering the contribution of the environment to protein structuring. The application of the fuzzy oil drop (FOD) model to assess the validity of the selected models provided in the CASP13, CASP14 and CASP15 projects reveals the need for an environmental factor to determine the 3D structure of proteins. Consideration of the external force field in the form of polar water (Fuzzy Oil Drop) and a version modified by the presence of the hydrophobic compounds, FOD-M (FOD-Modified) reveals that the protein folding process is environmentally dependent. An analysis of selected models from the CASP competitions indicates the need for structure prediction as dependent on the consideration of the protein folding environment.
CONCLUSIONS
The conditions governed by the environment direct the protein folding process occurring in a certain environment. Therefore, the variation of the external force field should be taken into account in the models used in protein structure prediction.
Topics: Artificial Intelligence; Models, Molecular; Proteins; Protein Folding; Hydrophobic and Hydrophilic Interactions; Protein Conformation
PubMed: 37950210
DOI: 10.1186/s12859-023-05559-8 -
Annual Review of Biophysics Jul 2016In vitro, computational, and theoretical studies of protein folding have converged to paint a rich and complex energy landscape. This landscape is sensitively modulated...
In vitro, computational, and theoretical studies of protein folding have converged to paint a rich and complex energy landscape. This landscape is sensitively modulated by environmental conditions and subject to evolutionary pressure on protein function. Of these environments, none is more complex than the cell itself, where proteins function in the cytosol, in membranes, and in different compartments. A wide variety of kinetic and thermodynamics experiments, ranging from single-molecule studies to jump kinetics and from nuclear magnetic resonance to imaging on the microscope, have elucidated how protein energy landscapes facilitate folding and how they are subject to evolutionary constraints and environmental perturbation. Here we review some recent developments in the field and refer the reader to some original work and additional reviews that cover this broad topic in protein science.
Topics: Humans; Kinetics; Protein Conformation; Protein Folding; Proteins; Thermodynamics
PubMed: 27391927
DOI: 10.1146/annurev-biophys-062215-011236 -
Methods in Molecular Biology (Clifton,... 2022We present a detailed heuristic method to quantify the degree of local energetic frustration manifested by protein molecules. Current applications are realized in...
We present a detailed heuristic method to quantify the degree of local energetic frustration manifested by protein molecules. Current applications are realized in computational experiments where a protein structure is visualized highlighting the energetic conflicts or the concordance of the local interactions in that structure. Minimally frustrated linkages highlight the stable folding core of the molecule. Sites of high local frustration, in contrast, often indicate functionally relevant regions such as binding, active, or allosteric sites.
Topics: Models, Molecular; Protein Conformation; Protein Folding; Proteins; Thermodynamics
PubMed: 34845622
DOI: 10.1007/978-1-0716-1716-8_22 -
Current Protocols in Protein Science Nov 2014Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion... (Review)
Review
Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed, (2) the cell wall and outer membrane components are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine·HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein, which is then partially purified by gel filtration before folding; the protein is then folded and oxidized by simple dialysis against water. Thirdly, folding and purification of a fusion protein using metal-chelate affinity chromatography are described.
Topics: Animals; Escherichia coli; Guanidine; Humans; Inclusion Bodies; Protein Denaturation; Protein Refolding; Recombinant Proteins
PubMed: 25367010
DOI: 10.1002/0471140864.ps0605s78 -
Methods (San Diego, Calif.) Sep 2018Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large... (Review)
Review
Membrane proteins (MPs) are important pharmacological targets because of their involvement in many essential cellular processes whose dysfunction can lead to a large variety of diseases. A detailed knowledge of the structure of MPs and the molecular mechanisms of their activity is essential to the design of new therapeutic agents. However, studying MPs in vitro is challenging, because it generally implies their overexpression under a functional form, followed by their extraction from membranes and purification. Targeting an overexpressed MP to a membrane is often toxic and expression yields tend to be limited. One alternative is the formation of inclusion bodies (IBs) in the cytosol of the cell, from which MPs need then to be folded to their native conformation before structural and functional analysis can be contemplated. Folding MPs targeted to IBs is a difficult task. Specially designed amphipathic polymers called 'amphipols' (APols), which have been initially developed with the view of improving the stability of MPs in aqueous solutions compared to detergents, can be used to fold both α-helical and β-barrel MPs. APols represent an interesting novel amphipathic medium, in which high folding yields can be achieved. In this review, the properties of APol A8-35 and of the complexes they form with MPs are summarized. An overview of the most important studies reported so far using A8-35 to fold MPs is presented. Finally, from a practical point of view, a detailed description of the folding and trapping methods is given.
Topics: Membrane Proteins; Polymers; Propylamines; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Folding; Protein Stability
PubMed: 29678587
DOI: 10.1016/j.ymeth.2018.04.012 -
Science Advances May 2018Precise protein folding is essential for the survival of all cells, and protein misfolding causes a number of diseases that lack effective therapies, yet the general...
Precise protein folding is essential for the survival of all cells, and protein misfolding causes a number of diseases that lack effective therapies, yet the general principles governing protein folding in the cell remain poorly understood. In vivo, folding can begin cotranslationally and protein quality control at the ribosome is essential for cellular proteostasis. We directly characterize and compare the refolding and cotranslational folding trajectories of the protein HaloTag. We introduce new techniques for both measuring folding kinetics and detecting the conformations of partially folded intermediates during translation in real time. We find that, although translation does not affect the rate-limiting step of HaloTag folding, a key aggregation-prone intermediate observed during in vitro refolding experiments is no longer detectable. This rerouting of the folding pathway increases HaloTag's folding efficiency and may serve as a general chaperone-independent mechanism of quality control by the ribosome.
Topics: Kinetics; Models, Molecular; Protein Conformation; Protein Folding; Protein Refolding; Proteins
PubMed: 29854950
DOI: 10.1126/sciadv.aas9098 -
Cell Systems Jun 2021Folding a linear chain of amino acids into a three-dimensional protein is a complex physical process that ultimately confers an impressive range of diverse functions.... (Review)
Review
Folding a linear chain of amino acids into a three-dimensional protein is a complex physical process that ultimately confers an impressive range of diverse functions. Although recent advances have driven significant progress in predicting three-dimensional protein structures from sequence, proteins are not static molecules. Rather, they exist as complex conformational ensembles defined by energy landscapes spanning the space of sequence and conditions. Quantitatively mapping the physical parameters that dictate these landscapes and protein stability is therefore critical to develop models that are capable of predicting how mutations alter function of proteins in disease and informing the design of proteins with desired functions. Here, we review the approaches that are used to quantify protein stability at a variety of scales, from returning multiple thermodynamic and kinetic measurements for a single protein sequence to yielding indirect insights into folding across a vast sequence space. The physical parameters derived from these approaches will provide a foundation for models that extend beyond the structural prediction to capture the complexity of conformational ensembles and, ultimately, their function.
Topics: Kinetics; Protein Folding; Protein Stability; Proteins; Thermodynamics
PubMed: 34139165
DOI: 10.1016/j.cels.2021.05.009 -
Biomacromolecules Oct 2022Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely...
Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.
Topics: Alginates; Phosphoglycerate Kinase; Polymers; Protein Denaturation; Protein Folding; Protein Stability
PubMed: 36054903
DOI: 10.1021/acs.biomac.2c00297