Engineered genetic selection links in vivo protein folding and stability with asparagine-linked glycosylation

Predicting the structural consequences of site-specific glycosylation remains a major challenge due in part to the lack of convenient experimental tools for rapidly determining how glycosylation influences protein folding. To address this shortcoming, we developed a genetic selection that directly links the in vivo folding of asparagine-linked (N-linked) glycoproteins with antibiotic resistance. Using this assay, we identified three known or putative glycoproteins from Campylobacter jejuni (Peb3, CjaA, and Cj0610c) whose folding was significantly affected by N-glycosylation. We also used the genetic selection to isolate a glycoengineered variant of the Escherichia coli colicin E7 immunity protein (Im7) whose intracellular folding and stability were enhanced as a result of N-glycosylation. In addition to monitoring the effect of glycan attachment on protein folding in living cells, this strategy could easily be extended for optimizing protein folding in vivo and engineering glycosylation enzymes, pathways, and hosts for optimal performance. See accompanying commentary by Danielle Tullman-Ercek DOI: 10.1002/biot.201300319     

Prolyl isomerization: How significant for in vivo protein folding?

Suggestive but not decisive evidence indicates that in vivo peptide chain folding is completed in a time not much longer than that required for covalent peptide synthesis. Extrapolation of model peptide rates of the cis–trans prolyl isomerization leads to the prediction tht protein folding should be much slower than the apparent in vivo rates. On the assumption that rapid protein folding in vivo is the rule, three routes are suggested by which a protein undergoing biosynthesis can avoid a strongly slowed folding rate: (1) by a peptide chain-elongation process that adds only trans peptide bonds, follwed by a rapid folding process that incorporates them into a three-dimensional structure, raising the energy barrier to isomerization; (2) by folding to produce three dimensional structures that position prolyl residues largely in chain turns on the protein surface, where the residue may be either cis or trans without large effects on the protein structure and function; (3) prolyl cis–trans isomerization may be speeded by the formation of peptide loops.     

A rapid protein folding assay for the bacterial periplasm

An array of genetic screens and selections has been developed for reporting protein folding and solubility in the cytoplasm of living cells. However, there are currently no analogous folding assays for the bacterial periplasm, despite the significance of this compartment for the expression of recombinant proteins, especially those requiring important posttranslational modifications (e.g., disulfide bond formation). Here, we describe an engineered genetic selection for monitoring protein folding in the periplasmic compartment of Escherichia coli cells. In this approach, target proteins are sandwiched between an N‐terminal signal recognition particle (SRP)‐dependent signal peptide and a C‐terminal selectable marker, TEM‐1 β‐lactamase. The resulting chimeras are localized to the periplasmic space via the cotranslational SRP pathway. Using a panel of native and heterologous proteins, we demonstrate that the folding efficiency of various target proteins correlates directly with in vivo β‐lactamase activity and thus resistance to ampicillin. We also show that this reporter is useful for the discovery of extrinsic periplasmic factors (e.g., chaperones) that affect protein folding and for obtaining folding‐enhanced proteins via directed evolution. Collectively, these data demonstrate that our periplasmic folding reporter is a powerful tool for screening and engineering protein folding in a manner that does not require any structural or functional information about the target protein.     

Cunning Simplicity of a Hierarchical Folding

Abstract

A hierarchic scheme of protein folding does not solve the Levinthal paradox since it cannot provide a simultaneous explanation for major features observed for protein folding: (i) folding within non-astronomical time, (ii) independence of the native structure on large variations in the folding rates of given protein under different conditions, and (iii) co-existence, in a visible quantity, of only the native and the unfolded molecules during folding of moderate size (single-domain) proteins. On the contrary, a nucleation mechanism can account for all these major features simultaneously and resolves the Levinthal paradox.     

Protein <Emphasis Type="Italic">N</Emphasis>-glycosylation,protein folding,and protein quality control

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.     

Lysozyme Folded In Silico According to the Limited Conformational Sub-space

Abstract

The conformational sub-space oriented on early-stage protein folding is applied to lysozyme folding. The part of the Ramachandran map distinguished on the basis of a geometrical model of the polypeptide chain limited to the mutual orientation of the peptide bond planes is shown to deliver the initial structure of the polypeptide for the energy minimization procedure in the ab initio model of protein folding prediction. Two forms of energy minimization and molecular dynamics simulation procedures were applied to the assumed early-stage protein folding of lysozyme. One of them included the disulphide bond system and the other excluded it. The post-energy-minimization and post-dynamics structures were compared using RMS-D and non-bonding contact maps to estimate the degree of approach to the native, target structure of the protein molecule obtained using the limited conformational sub-space for the early stage of folding.     

Protein folding and chaperonins

The folding of polypeptide chains in cells, following either translation or translocation through membranes, must take place under conditions of extremely high protein concentrations. In addition, folding into a correct structure must occur in the presence of other rapidly folding species, and at temperatures known to destabilize aggregation-prone folding intermediates. To facilitate folding in vivo, molecular chaperones have evolved that stabilize protein folding intermediates, thus partitioning them towards a pathway leading to the native state rather than forming inactive aggregated structures.     

Recent Advances in GFP Folding Reporter and Split-GFP Solubility Reporter Technologies. Application to Improving the Folding and Solubility of Recalcitrant Proteins from Mycobacterium tuberculosis

We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691–695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102–107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.     

Polytopic membrane protein folding and assembly in vitro and in vivo (Review)

The insertion and folding of proteins in biological membranes during protein synthesis in vivo is fundamental to membrane biogenesis. At present, however, certain molecular aspects of this process can only be understood by complementary studies in vitro. We bring together in vitro and in vivo results, highlighting how the studies inform each other and increase our knowledge of the folding and assembly of polytopic membrane proteins. A notable recent advance is the high-resolution crystal structure of the protein machinery responsible for membrane protein insertion into the endoplasmic reticulum. This provides an opportunity to combine in vitro and in vivo studies at a more sophisticated level and address mechanistic aspects of polytopic protein insertion and folding. Quality control is another important aspect of membrane biogenesis, and we give an overview of the current understanding of this process, focusing on cystic fibrosis as a well-studied paradigm. Mutations in the associated membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), can cause the quality control mechanisms to prevent the mutant protein reaching its normal site of action, the cell surface. In vitro studies of CFTR shed light on the possible origins of other clinically relevant folding mutants and highlight the potential synergy between in vitro and in vivo approaches.     

A comprehensive database of verified experimental data on protein folding kinetics

Insights into protein folding rely increasingly on the synergy between experimental and theoretical approaches. Developing successful computational models requires access to experimental data of sufficient quantity and high quality. We compiled folding rate constants for what initially appeared to be 184 proteins from 15 published collections/web databases. To generate the highest confidence in the dataset, we verified the reported lnk_f value and exact experimental construct and conditions from the original experimental report(s). The resulting comprehensive database of 126 verified entries, ACPro, will serve as a freely accessible resource ( https://www.ats.amherst.edu/protein/ ) for the protein folding community to enable confident testing of predictive models. In addition, we provide a streamlined submission form for researchers to add new folding kinetics results, requiring specification of all the relevant experimental information according to the standards proposed in 2005 by the protein folding consortium organized by Plaxco. As the number and diversity of proteins whose folding kinetics are studied expands, our curated database will enable efficient and confident incorporation of new experimental results into a standardized collection. This database will support a more robust symbiosis between experiment and theory, leading ultimately to more rapid and accurate insights into protein folding, stability, and dynamics.     

Identification and characterization of folding inhibitors of hen egg lysozyme: an example of a new paradigm of drug design

Studies of protein folding indicate the presence of native contacts in the denatured state, giving rise to folding elements which contribute to the accomplishment of the native state. The possibility of finding molecules which can interact with specific folding elements of a target protein preventing it from reaching its native state, and hence from becoming biologically active, is particularly attractive. The notion that folding elements not only provide molecular recognition directing the folding process, but also have conserved sequence, implies that targeting such elements will make protein folding inhibitors less susceptible to mutations which, in many cases, abrogate drug effects. The folding-inhibition strategy can lead to a truly novel and rational approach to drug design, aside from providing new insight into folding. This is illustrated in the case of hen egg lysozyme.     

Chaperonin 60: a paradoxical,evolutionarily conserved protein family with multiple moonlighting functions

Chaperonin 60 is the prototypic molecular chaperone, an essential protein in eukaryotes and prokaryotes, whose sequence conservation provides an excellent basis for phylogenetic analysis. Escherichia coli chaperonin 60 (GroEL), the prototype of this family of proteins, has an established oligomeric‐structure‐based folding mechanism and a defined population of folding partners. However, there is a growing number of examples of chaperonin 60 proteins whose crystal structures and oligomeric composition are at variance with GroEL, suggesting that additional complexities in the protein‐folding function of this protein should be expected. In addition, many organisms have multiple chaperonin 60 proteins, some of which have lost their protein‐folding ability. It is emerging that this highly conserved protein has evolved a bewildering variety of additional biological functions – known as moonlighting functions – both within the cell and in the extracellular milieu. Indeed, in some organisms, it is these moonlighting functions that have been left after the loss of the protein‐folding activity. This highlights the major paradox in the biology of chaperonin 60. This article reviews the relationship between the folding and non‐folding (moonlighting) activities of the chaperonin 60 family and discusses current knowledge on their molecular evolution focusing on protein domains involved in the non‐folding chaperonin functions in an attempt to understand the emerging biology of this evolutionarily ancient protein family.     

The stability of Taq DNA polymerase results from a reduced entropic folding penalty; identification of other thermophilic proteins with similar folding thermodynamics

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs‐Helmholtz stability curves of the free energy of folding (ΔG) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (ΔH and ΔS), and the heat capacity (ΔC_p) of folding. If increased or enhanced non‐covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow–Klentaq homologous pair, the folding enthalpy (ΔH_fold) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq's extreme free energy of folding (ΔG_fold) originates from a significantly reduced entropic penalty of folding (ΔS_fold). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic–thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar “reduced ΔC_p mechanism” for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced‐entropic‐penalty model. Proteins 2014; 82:785–793. © 2013 Wiley Periodicals, Inc.     

Monitoring protein stability <Emphasis Type="Italic">in vivo</Emphasis>

Reduced protein stability in vivo is a prerequisite to aggregation. While this is merely a nuisance factor in recombinant protein production, it holds a serious impact for man. This review focuses on specific approaches to selectively determine the solubility and/or stability of a target protein within the complex cellular environment using different detection techniques. Noninvasive techniques mapping folding/misfolding events on a fast time scale can be used to unravel the complexity and dynamics of the protein aggregation process and factors altering protein solubility in vivo. The development of approaches to screen for folding and solubility in vivo should facilitate the identification of potential components that improve protein solubility and/or modulate misfolding and aggregation and may provide a therapeutic benefit.     

GroEL的耗能分子伴侣机制

双环结构Gro EL及其辅分子伴侣Gro ES是目前研究得最深入的分子伴侣.然而,Gro EL/Gro ES帮助蛋白质折叠的一些关键理化机制,尤其是水解ATP,Gro EL发生构象改变,能否主动调节蛋白质错误折叠中间体的构象,以促进错误折叠中间体的复性,仍然存在争议.结合本研究组近年的工作,作者着力介绍Gro EL促进蛋白质折叠的主动解折叠机制.     

Back to Units of Protein Folding

Abstract

In response to the criticism by A. Finkelstein (J. Biomol. Struct. Dyn. 20, 311–314, 2002) of our Communication (J. Biomol. Struct. Dyn. 20, 5–6, 2002) several issues are dealt with. Importance of the notion of elementary folding unit, its size and structure, and the necessity of further characterization of the units for the elucidation of the protein folding in vivo are discussed. The criticism (J. Biomol. Struct. Dyn. 20, 311–314, 2002) on the hierarchical protein folding is also briefly addressed.     

GB1 is not a two-state folder: identification and characterization of an on-pathway intermediate

The folding pathway of the small α/β protein GB1 has been extensively studied during the past two decades using both theoretical and experimental approaches. These studies provided a consensus view that the protein folds in a two-state manner. Here, we reassessed the folding of GB1, both by experiments and simulations, and detected the presence of an on-pathway intermediate. This intermediate has eluded earlier experimental characterization and is distinct from the collapsed state previously identified using ultrarapid mixing. Failure to identify the presence of an intermediate affects some of the conclusions that have been drawn for GB1, a popular model for protein folding studies.     

The chaperonin cycle and protein folding

The process of protein folding in the cell is now known to depend on the action of other proteins. These proteins include molecular chaperones, Which interact non-covalently with proteins as they fold and improve the final yields of active protein in the cell. The precise mechanism by which molecular chaperones act is obscure. Experiments reported recently⁽¹⁾ show that for one molecular chaperone (Cpn60, typified by the E. coli protein GroEL), the folding reaction is driven by cycles of binding and release of the co-chaperone Cpn10 (known as GroES in E. coli). These alternate with binding and release of the unfolded protein substrate. These cycles come about because of the opposite effects of Cpn10 and unfolded protein on the Cpn60 complex: the former stabilises the ADP-bound state of Cpn60, whereas the latter stimulates ADP-ATP exchange. This model proposes that the substrate protein goes through multiple cycles of binding and release, and is released into the cavity of the Cpn60 complex where it can undergo folding without interacting with other nearby folding intermediates. This is consistent with the ability of Cpn60 proteins to enhance folding by blocking pathways to aggregation.     

A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

Recombinant expression of native or modified eukaryotic proteins is pivotal for structural and functional studies and for industrial and pharmaceutical production of proteins. However, it is often impeded by the lack of proper folding. Here, we present a stringent and broadly applicable eukaryotic in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C‐terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality‐control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully demonstrated using a complex insertion mutant library of TNF‐α, from which different folding competent mutant proteins were uncovered.