Native-like Properties Research Articles

Making a stabilized HLA-E complex with native properties.

Abstract Objective The non-classical, human MHC-I molecule HLA-E is functionally monomorphic and therefore a highly attractive target in immunotherapy against cancers and infectious diseases. However, HLA-E is characterized by low stability due to its biological role as a rapid-response system to monitor HLA-I expression. The inherent instability makes production of HLA-E difficult and severely limits its use as a reagent, which in turn negatively impacts the understanding of HLA-E biology. Here, we present native-like, stabilized forms of HLA-E, which brings production of HLA-E in line with that of classical MHC-I molecules. Methods Disulfide bridges were introduced in HLA-E at selected locations deemed to not interfere with recognition of HLA-E by NK or T cell receptors. The stability of the modified HLA-E molecules was monitored using thermal denaturation, and conservation of native HLA-E properties was assessed by in vitro TCR-binding assays and through staining of NK cells. Results The modified HLA-E molecules exhibited increased stability, increased refolding yields, both or neither. The most stable HLA-E variant exhibited thermal stability comparable to complexes of HLA-A*02:01, while retaining native-like recognition by both NK and T cell receptors. Summary Stabilized forms of HLA-E have been generated and their native-like properties validated using in vitro assays and NK cell staining. This enables production of reagents for the study HLA-E biology.

Optimizing the Production of gp145, an HIV-1 Envelope Glycoprotein Vaccine Candidate and Its Encapsulation in Guanosine Microparticles.

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties.

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.

EGFR extracellular domain III expressed in Escherichia coli with SEP tag shows improved biophysical and functional properties and generate anti-sera inhibiting cancer cell growth

The epidermal growth factor receptor extracellular domain III (EGFR-ECDIII) protein is a promising target of anti-cancer research, and its production in Escherichia coli would thus represent significant benefits. However, despite its moderate size (19 kDa), the expression of EGFR-ECDIII in E.coli is hampered by the presence of multiple cysteines producing misfolded proteins with incorrect S-S bonds. In our study, we show that a short 12-residue solubility enhancing peptide (SEP) tag containing nine arginines (C9R) attached at the C-terminus of EGFR-ECDIII reduces the inclusion body formation and increases the final yield by six times (20 mg/L). EGFR-ECDIII-C9R purified from the soluble fraction eluted as a sharp single RP-HPLC peak, suggesting a single S-S bond pairing. Biophysical characterization using circular dichroism, fluorescence, and light scattering confirmed its native-like properties together with reversible thermal denaturation. The binding activity of EGFR-ECDIII-C9R to anti-EGFR-VHH7D12, a single-domain antibody with specific binding to the ECDIII, was assessed by sandwich ELISA. Further, we produced anti-EGFR-ECDIII-C9R antisera in mouse models and anti-sera inhibited A431 cancer cells’ growth. These results demonstrate that the SEP tag enables the rapid production of the multiple disulfide-bonded EGFR-ECDIII in E. coli having native-like biophysical properties and producing neutralizing antibodies.

Effect of centrifugal force on the development of articular neocartilage with bovine primary chondrocytes

A lot has been invested into understanding how to assemble cartilage tissue in vitro and various designs have been developed to manufacture cartilage tissue with native-like biological properties. So far, no satisfactory design has been presented. Bovine primary chondrocytes are used to self-assemble scaffold-free constructs to investigate whether mechanical loading by centrifugal force would be useful in manufacturing cartilage tissue in vitro. Six million chondrocytes were laid on top of defatted bone disks placed inside an agarose well in 50-ml culture tubes. The constructs were centrifuged once or three times per day for 15 min at a centrifugal force of 771×g for up to 4 weeks. Control samples were cultured under the same conditions without exposure to centrifugation. The samples were analysed by (immuno)histochemistry, Fourier transform infrared imaging, micro-computed tomography, biochemical and gene expression analyses. Biomechanical testing was also performed. The centrifuged tissues had a more even surface covering a larger area of the bone disk. Fourier transform infrared imaging analysis indicated a higher concentration of collagen in the top and bottom edges in some of the centrifuged samples. Glycosaminoglycan contents increased along the culture, while collagen content remained at a rather constant level. Aggrecan and procollagen α1(II) gene expression levels had no significant differences, while procollagen α2(I) levels were increased significantly. Biomechanical analyses did not reveal remarkable changes. The centrifugation regimes lead to more uniform tissue constructs, whereas improved biological properties of the native tissue could not be obtained by centrifugation.

Mussel Byssus Structure-Function and Fabrication as Inspiration for Biotechnological Production of Advanced Materials.

Biotechnology offers an exciting avenue toward the sustainable production of high performance proteinaceous polymeric materials. In particular, the mussel byssus-a high performance adhesive bio-fiber used by mussels to cling on hard surfaces-has become a veritable archetype for bio-inspired self-healing fibers, tough coatings, and versatile wet adhesives. However, successful translation of mussel-inspired design principles into man-made materials hinges upon elucidating structure-function relationships and biological fabrication processes. This review provides a detailed survey of the state-of-the-art understanding of the biochemical structure-function relationships defining byssus performance with a particular focus on structural hierarchy and metal coordination-based cross-linking. The efforts to mimic the byssus in man-made materials are then discussed. While there has been a strong push to mimic the byssus via synthetic chemistry taking a reductionist approach, herein the focus is specifically on recent progress of biotechnology-based strategies that more closely approximate the biochemical complexity of the natural material. As an outlook, an overview of recent research toward understanding the natural byssus assembly process is provided, as processing remains a critical factor in achieving native-like properties.

Direct Conversion of an Enzyme from Native-like to Amyloid-like Aggregates within Inclusion Bodies

The acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli. Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into β-sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state.

Proteolytic cleavage of the hydrophobic domain in the CaVα2δ1 subunit improves assembly and activity of cardiac CaV1.2 channels

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVβ, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.

Statistical Mechanical Model for pH-Induced Protein Folding: Application to Apomyoglobin.

Despite the major role of pH in protein folding and stability, a quantitative understanding of the pH-induced protein folding mechanism remains elusive. Two conventional models, the Monod-Wyman-Changeux and Linderstrøm-Lang smeared charge models, respectively, have been used to analyze the formation/disruption of specific native structures and fluctuating non-native states. However, there are only a few models that can represent the overall kinetic events of folding/unfolding independent of the properties of relevant molecular species, which has hampered the efforts to systematically analyze pH-induced folding. Here, we constructed a statistical mechanical model that incorporates the protonation mechanism of conventional models along with a combined manual search and least-squares fitting procedure, which was used to investigate the folding of horse apomyoglobin over a wide pH range (2.2-6.7), with a time window ranging from ∼40 μs to ∼100 s, using continuous-/stopped-flow fluorescence at 8 °C. Quantitative analysis assuming a five-state sequential scheme indicated that (1) pH-induced folding/unfolding is represented by both specific binding and Coulombic interactions; (2) kinetic folding/unfolding intermediates share kinetic mechanisms with the equilibrium intermediate, indicating their equivalence; and (3) native-like properties are acquired successively during folding by intermediates and in transition states. This model could also be applied to a variety of association/dissociation processes.

A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina.

BackgroundOne of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence.ResultsThe constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity.ConclusionsThe elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.

Well-ordered trimeric HIV-1 subtype B and C soluble spike mimetics generated by negative selection display native-like properties.

The structure of BG505 gp140 SOSIP, a soluble mimic of the native HIV-1 envelope glycoprotein (Env), marks the beginning of new era in Env structure-based immunogen design. Displaying a well-ordered quaternary structure, these subtype A-derived trimers display an excellent antigenic profile, discriminating recognition by broadly neutralizing antibodies (bNAbs) from non-broadly neutralizing antibodies (non-bNAbs), and provide a solid Env-based immunogenic platform starting point. Even with this important advance, obtaining homogeneous well-ordered soluble SOSIP trimers derived from other subtypes remains challenging. Here, we report the “rescue” of homogeneous well-ordered subtype B and C SOSIP trimers from a heterogeneous Env mixture using CD4 binding site-directed (CD4bs) non-bNAbs in a negative-selection purification process. These non-bNAbs recognize the primary receptor CD4bs only on disordered trimers but not on the native Env spike or well-ordered soluble trimers due to steric hindrance. Following negative selection to remove disordered oligomers, we demonstrated recovery of well-ordered, homogeneous trimers by electron microscopy (EM). We obtained 3D EM reconstructions of unliganded trimers, as well as in complex with sCD4, a panel of CD4bs-directed bNAbs, and the cleavage-dependent, trimer-specific bNAb, PGT151. Using bio-layer light interferometry (BLI) we demonstrated that the well-ordered trimers were efficiently recognized by bNAbs and poorly recognized by non-bNAbs, representing soluble mimics of the native viral spike. Biophysical characterization was consistent with the thermostability of a homogeneous species that could be further stabilized by specific bNAbs. This study revealed that Env trimers generate different frequencies of well-ordered versus disordered aberrant trimers even when they are genetically identical. By negatively selecting the native-like well-ordered trimers, we establish a new means to obtain soluble Env mimetics derived from subtypes B and C for expanded use as candidate vaccine immunogens.

The Effect of Varying Concentrations and Application Periods of Chondroitinase ABC on Tissue-Engineered Cartilage

Osteoarthritis, a chronic malady characterized by joint pain and swelling, is caused by damage to articular cartilage and is perpetuated by low-grade inflammation. Treatments for osteoarthritis do exist, but many treatments focus on coping with the disease rather than curing it. Surgical options that replace damaged cartilage tissue with that of donor cartilage tissue or cartilage tissue from other parts of articular joints face complications especially when the tissue is not of the correct size or does not have native-like properties.
 A more suitable treatment option for osteoarthritis is to develop an in vitro tissue-engineered cartilage construct that can be grown using the patient’s own cells and to surgically remove the patient’s damaged cartilage and replace it with the tissue-engineered cartilage. A challenge in developing such a treatment option is producing tissue-engineered cartilage with mechanical properties akin to those of native human articular cartilage. This challenge may be overcome by maximizing the production of type II collagen by the chondrocytes in vitro. One way to maximize collagen production is through the application of chondroitinase ABC, an enzyme which temporarily suppresses proteoglycans in the cartilage matrix to create more space for type II collagen to develop.
 In this study, two two levels of cABC treatment were applied (“high” and “low”) to cartilage tissue constructs. The “low” cABC treated group received daily feeding of 0.075 U/mL from day 14 to 21 followed by a replacement of chondrogenic media without cABC. The “high” cABC treated group received a single addition of 0.15 U/mL from day 14 to 16 followed by a replacement of chondrogenic media without cABC. At the end of 42 days, the constructs were subjected to mechanical testing and biochemical analyses. These analyses showed that the high cABC treatment yielded more native-like mechanical properties when compared to the low cABC treatment and the control results. Biochemical and histological analyses confirmed that the proteoglycan and collagen II content were higher in the low and high cABC treated groups when compared to the control. All analyses show that the most efficient application of chondroitinase ABC is through a two day duration treatment of a higher concentration (0.15 U/mL).

Physicochemical Studies of Bacteriorhodopsin Reconstituted in Partially Fluorinated Phosphatidylcholine Bilayers

A membrane protein bacteriorhodopsin (bR) that is successfully reconstituted in liposome of a novel partially fluorinated analog of dimyristoylphosphatidylcholine (DMPC) with the perfluorobutyl segments in the myristoyl groups, diF4H10-PC, has been investigated by some spectroscopic and X-ray diffraction techniques to clarify effects of substitution of nine hydrogen atoms by fluorine atoms on structural and physical properties of the membrane protein by comparison with the previous results on proteoliposome of bR and DMPC. Below the gel-to-liquid crystalline phase transition of diF4H10-PC bilayer, bR molecules adopt the two-dimensional lattice structure of trimers as the structural unit and show a photocycle very similar to that of native purple membrane like reconstituted bR in DMPC liposome in the gel phase. Even upon heating up to temperatures well above the phase transition, the nativelike functional reconstitution and higher structural stability of bR molecules in diF4H10-PC liposome are retained, which strikingly contrasts with lipid phase transition-induced disaggregation of protein molecules and light-induced denaturation in DMPC liposome. Greater membrane rigidity and low affinity between bR and fluorinated lipid molecules are proposed as a driving force for keeping nativelike properties of bR molecules in diF4H10-PC liposome even in the fluid phase.

Triosephosphate Isomerase by Consensus Design: Dramatic Differences in Physical Properties and Activity of Related Variants

Consensus design, the selection of mutations based on the most common amino acid in each position of a multiple sequence alignment, has proven to be an efficient way to engineer stabilized mutants and even to design entire proteins. However, its application has been limited to small motifs or small families of highly related proteins. Also, we have little idea of how information that specifies a protein's properties is distributed between positional effects (consensus) and interactions between positions (correlated occurrences of amino acids). Here, we designed several consensus variants of triosephosphate isomerase (TIM), a large, diverse family of complex enzymes. The first variant was only weakly active, had molten globular characteristics, and was monomeric at 25 °C despite being based on nearly all dimeric enzymes. A closely related variant from curation of the sequence database resulted in a native-like dimeric TIM with near-diffusion-controlled kinetics. Both enzymes vary substantially (30–40%) from any natural TIM, but they differ from each other in only a relatively small number of unconserved positions. We demonstrate that consensus design is sufficient to engineer a sophisticated protein that requires precise substrate positioning and coordinated loop motion. The difference in oligomeric states and native-like properties for the two consensus variants is not a result of defects in the dimerization interface but rather disparate global properties of the proteins. These results have important implications for the role of correlated amino acids, the ability of TIM to function as a monomer, and the ability of molten globular proteins to carry out complex reactions.

Absolute quality evaluation of protein model structures using statistical potentials with respect to the native and reference states

In protein structure prediction, it is crucial to evaluate the degree of native-likeness of given model structures. Statistical potentials extracted from protein structure data sets are widely used for such quality assessment problems, but they are only applicable for comparing different models of the same protein. Although various other methods, such as machine learning approaches, were developed to predict the absolute similarity of model structures to the native ones, they required a set of decoy structures in addition to the model structures. In this paper, we tried to reformulate the statistical potentials as absolute quality scores, without using the information from decoy structures. For this purpose, we regarded the native state and the reference state, which are necessary components of statistical potentials, as the good and bad standard states, respectively, and first showed that the statistical potentials can be regarded as the state functions, which relate a model structure to the native and reference states. Then, we proposed a standardized measure of protein structure, called native-likeness, by interpolating the score of a model structure between the native and reference state scores defined for each protein. The native-likeness correlated with the similarity to the native structures and discriminated the native structures from the models, with better accuracy than the raw score. Our results show that statistical potentials can quantify the native-like properties of protein structures, if they fully utilize the statistical information obtained from the data set.

Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase

BackgroundChitin synthase 3a (CHS3a) from Botrytis cinerea (Bc) catalyses the multiple transfer of N-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a β-1,4 linked GlcNAc homopolymer, is an essential cell wall component of filamentous fungi. Chitin synthase, processive membranous protein, has been recognized as a promising target for new antifungicides. Enzymatic characterizations of chitin synthases have been limited, mainly because purity and amounts of integral enzyme obtained after purification procedures have not been sufficient.FindingsWe undertook the preparation of two BcCHS3a fragment proteins, containing only the central domain and devoid of the N-terminal and transmembrane C-terminal regions. The central domain of CHS3a, named SGC (Spsa GntI Core), is conserved in all UDP-glycosyltransferases and it is believed to contain the active site of the enzyme. CHS3a-SGC protein was totally expressed as inclusion bodies in Escherichia coli. We performed recombinant CHS3a-SGC purification in denaturing conditions, followed by a refolding step. Although circular dichroism spectra clearly exhibited secondary structures of renatured CHS3a-SGC, no chitin synthase activity was detected. Nevertheless CHS3a-SGC proteins show specific binding for the substrate UDP-GlcNAc with a dissociation constant similar to the Michaelis constant and a major contribution of the uracil moiety for recognition was confirmed.ConclusionsMilligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be easily obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a.

Rapid Collapse into a Molten Globule Is Followed by Simple Two-State Kinetics in the Folding of Lysozyme from Bacteriophage λ

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage λ (λ lysozyme) at pH 5.6, 20 °C. The first step in the folding of λ lysozyme occurs very rapidly (τ < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of secondary structure but only weak protection from (1)H/(2)H exchange and no fixed tertiary structure organization. This early folding step is reflected in the dead-time events observed in the far-UV CD and ANS fluorescence experiments. Following accumulation of this kinetic molten globule species, the secondary structural elements are stabilized and the majority (ca. 88%) of refolding molecules acquire native-like properties in a highly cooperative two-state process, with τ = 0.15 ± 0.03 s. This is accompanied by the acquisition of substantial native-like protection from hydrogen exchange. A double-mixing experiment and the absence of a denaturant effect reveal that slow (τ = 5 ± 1 s) folding of the remaining (ca. 12%) molecules is rate limited by the cis/trans isomerization of prolines that are trans in the folded enzyme. In addition, native state hydrogen exchange and classical denaturant unfolding experiments have been used to characterize the thermodynamic properties of the enzyme. In good agreement with previous crystallographic evidence, our results show that λ lysozyme is a highly dynamic protein, with relatively low conformational stability (ΔG°(N-U) = 25 ± 2 kJ·mol(-1)).

Lysophospholipid Micelles Sustain the Stability and Catalytic Activity of Diacylglycerol Kinase in the Absence of Lipids

There has been a renewal of interest in interactions of membrane proteins with detergents and lipids, sparked both by recent results that illuminate the structural details of these interactions and also by the realization that some experimental membrane protein structures are distorted by detergent-protein interactions. The integral membrane enzyme diacylglycerol kinase (DAGK) has long been thought to require the presence of lipid as an obligate "cofactor" in order to be catalytically viable in micelles. Here, we report that near-optimal catalytic properties are observed for DAGK in micelles composed of lysomyristoylphosphatidylcholine (LMPC), with significant activity also being observed in micelles composed of lysomyristoylphosphatidylglycerol and tetradecylphosphocholine. All three of these detergents were also sustained high stability of the enzyme. NMR measurements revealed significant differences in DAGK-detergent interactions involving LMPC micelles versus micelles composed of dodecylphosphocholine. These results highlight the fact that some integral membrane proteins can maintain native-like properties in lipid-free detergent micelles and also suggest that C(14)-based detergents may be worthy of more widespread use in studies of membrane proteins.

Computational studies of the structure, dynamics and native content of amyloid‐like fibrils of ribonuclease A

The characterization at atomic resolution of amyloid-like protein aggregates is one of the fundamental problems of modern biology. In particular, the question whether native-like domains are retained or completely refolded in the amyloid state and the identification of possible mechanisms for macromolecular ordered aggregation represent major unresolved puzzles. To address these issues, in this article we examine the stability, dynamics, and conservation of native-like properties of several models of a previously designed amyloid-like fibril of RNase A (Sambashivan et al., Nature 2005; 437:266-269). Through the use of molecular dynamics (MD) simulations, we have provided molecular-level insights into the role of different parts of the sequence on the stability of fibrils, the collective properties of supramolecular complexes, and the presence of native-like conformations and dynamics in supramolecular aggregates. We have been able to show that within the fibrils the three-dimensional globular domain-swapped units preserve the conformational, dynamical, and hydration properties typical of the monomeric state, providing a rationalization for the experimentally observed catalytic activity of fibrils. The nativeness of the globular domains is not affected by the amyloidogenic stretches, which determine the molecular recognition process underlying aggregation through the formation of a stable steric zipper motif. Moreover, through the study of the hydration features of a single sheet model, we have been able to show that polyglutamine stretches of the domain-swapped ribonuclease tend to minimize the interaction with water in favor of sidechain-sidechain interactions, shedding light on the factors leading to the supramolecular assembly of beta-sheet layers into dry steric zippers.

An investigation into the native‐like properties of de novo designed cavitand‐based four‐helix bundle proteins

We investigated the hydrophobic packing of two previously designed caviteins, LG2 and LG3, which differ by one Gly in the linker regions between the peptide sequence and the cavitand scaffold. We sought to diminish the putative native-like properties of LG2 and LG3, and see if we could diagnose a change in the conformational specificity of the hydrophobic core. We replaced the leucine residues with norleucine residues at the hydrophobic positions in LG2 and LG3, to create NG2 and NG3, respectively. LG2 exhibited more dispersion, but less sharp signals than LG3 in the amide region of its (1)H NMR spectrum. NG3 and NG2 were found to be slightly less helical and significantly less stable toward guanidine hydrochloride compared with their reference caviteins. The (1)H NMR spectrum of NG2 was very similar to that of LG2, whereas there was a noticeable loss in the number and sharpness of the amide signals of NG3 compared with LG3. These data suggest that LG3 is very well packed; a loss in conformational specificity resulted from replacement of the leucine residues with norleucine residues. In contrast, the packing and dynamics of the hydrophobic core in LG2 were similar to those in NG2 (both more modest than LG3), as their (1)H NMR spectra were virtually indistinguishable. Overall, substitution of leucine by norleucine provided an efficient, convenient, and informative probe of the packing and dynamics of our caviteins' hydrophobic cores.