Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion under denaturing conditions, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of the formerly N-glycosylated peptides. LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases.

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Osmolytes as Modulators of Conformational Changes in Serpins

Biological Chemistry ◽

10.1515/bc.2001.194 ◽

2001 ◽

Vol 382 (11) ◽

pp. 1593-1599 ◽

Cited By ~ 19

Author(s):

Michelle K.M. Chow ◽

Glyn L. Devlin ◽

Stephen P. Bottomley

Keyword(s):

Conformational Changes ◽

Protein Misfolding ◽

Structural Changes ◽

Serine Proteinase ◽

Thermally Induced ◽

Naturally Occurring ◽

Antitrypsin Deficiency ◽

Integral Role ◽

Protein Misfolding And Aggregation ◽

Insoluble Polymers

Abstract Protein misfolding and aggregation play an integral role in many diseases. The misfolding of the serpin (SERine Proteinase INhibitor) α1-antitrypsin results in the accumulation of insoluble polymers within hepatocytes and α1-antitrypsin deficiency in plasma, predisposing patients to liver cirrhosis and emphysema. We have examined the effect of three naturally occurring osmolytes, sarcosine, glycine betaine and trimethylamine Noxide, on conformational changes in α1-antitrypsin. All three solutes protected native α1-antitrypsin against thermally induced polymerisation and inactivation in a concentrationdependent manner. Further spectroscopic analysis showed that sarcosine stabilises the native conformation of α1-antitrypsin, thus hindering its conversion to an intermediate state and subsequent polymerisation. On refolding in the presence of sarcosine, α1-antitrypsin formed a heterogeneous population, with increasing proportions of molecules adopting an inactive conformation in higher concentrations of the osmolyte. These data show that sarcosine can be used to prevent abnormal structural changes in native α1-antitrypsin, but is ineffective in facilitating the correct folding of the protein. The implications of these results in the context of conformational changes and states adopted by α1-antitrypsin are discussed.

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Structural changes of a protein extract from apple with polyphenoloxidase activity obtained by cationic reversed micellar extraction induced by high-pressure carbon dioxide and thermosonication

Scientific Reports ◽

10.1038/s41598-019-50209-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

A. E. Illera ◽

S. Beltrán ◽

M. T. Sanz

Keyword(s):

Carbon Dioxide ◽

Conformational Changes ◽

Structural Changes ◽

Ammonium Bromide ◽

Protein Extract ◽

Experimental Conditions ◽

Micellar Extraction ◽

Activity Recovery ◽

Good Technique ◽

Triton X

Abstract Polyphenoloxidase from apple was extracted and further concentrated by cationic reversed micellar extraction. Previous to reversed micellar extraction a crude protein extract was obtained using AG2-X8 as adsorbent of phenolic compounds and the detergent Triton X-100. Forward and backward extraction conditions were optimized by using dodecyl trimethyl ammonium bromide as surfactant in the organic phase. Optimization was carried out to obtain the highest value of PPO activity recovery and the purification fold at the different experimental conditions. Under the optimum extraction conditions, PPO activity recovery was 99% and purification fold reached a value of 17, showing that reversed micellar extraction was a good technique as a first step to concentrate on a targeted enzyme. After removing some impurities by centrifuge ultrafiltration, the protein extract with PPO activity was treated by pressurized carbon dioxide and thermosonication achieving residual PPO activity values of 16 ± 3 and 9 ± 1%, respectively. Quenching experiments by iodide performed in the non-treated extract and in the treated extracts revealed conformational changes of this protein fraction reflected in the greater exposure of the fluorophore to the quencher.

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Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128833 ◽

1987 ◽

Vol 45 ◽

pp. 910-911

Author(s):

M. Boublik ◽

V. Mandiyan ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

16S Rrna ◽

Conformational Changes ◽

Ribosomal Proteins ◽

Structural Changes ◽

Ribosomal Subunit ◽

Compact Structure ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques

Protein and Peptide Letters ◽

10.2174/0929866526666191002113525 ◽

2020 ◽

Vol 27 (3) ◽

pp. 201-209

Author(s):

Syed Saqib Ali ◽

Mohammad Khalid Zia ◽

Tooba Siddiqui ◽

Haseeb Ahsan ◽

Fahim Halim Khan

Keyword(s):

Ascorbic Acid ◽

Conformational Changes ◽

Structural Changes ◽

The Body ◽

Titration Calorimetry ◽

Spectroscopic Techniques ◽

Human Beings ◽

Plasma Ascorbic Acid ◽

Dietary Antioxidant ◽

And Function

Background: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. Objective: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep’s blood. Methods: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. Conclusion: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

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Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles

Molecules ◽

10.3390/molecules26123607 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3607

Author(s):

Olena Dobrovolska ◽

Øyvind Strømland ◽

Ørjan Sele Handegård ◽

Martin Jakubec ◽

Morten L. Govasli ◽

...

Keyword(s):

Conformational Changes ◽

Protein Misfolding ◽

Driving Forces ◽

Conformational Space ◽

Supported Bilayers ◽

Conformational Ensembles ◽

Protein Membrane ◽

Chimeric Polypeptide ◽

Protein Misfolding And Aggregation ◽

In Silico Analyses

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.

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Metastability in Monolayer Films Transferred onto Solid Substrates by the Langmuir-Blodgett Method: IR Evidence for Transfer-Induced Phase Transitions

Applied Spectroscopy ◽

10.1366/0003702944027309 ◽

1994 ◽

Vol 48 (10) ◽

pp. 1196-1203 ◽

Cited By ~ 18

Author(s):

Fazale R. Rana ◽

Suci Widayati ◽

Brian W. Gregory ◽

Richard A. Dluhy

Keyword(s):

Fatty Acid ◽

Conformational Changes ◽

Structural Changes ◽

High Surface ◽

Solid Substrate ◽

Water Interface ◽

Monolayer Films ◽

Langmuir Blodgett ◽

Monomolecular Film ◽

Air Water Interface

The rate at which a monomolecular film is deposited onto a solid substrate in the Langmuir-Blodgett process of preparing supported monolayer films influences the final structure of the transferred film. Attenuated total reflectance infrared spectroscopic studies of monolayers transferred to germanium substrates show that the speed at which the substrate is drawn through the air/water interface influences the final conformation in the hydrocarbon chains of amphiphilic film molecules. This transfer-induced effect is especially evident when the monolayer is transferred from the expanded region of surface-pressure-molecular-area isotherms at low surface pressures; the effect is minimized when the film molecules are transferred from condensed phases at high surface pressures. This phenomenon has been observed for both a fatty acid and a phospholipid, which suggests that these conformational changes may occur in a variety of hydrocarbon amphiphiles transferred from the air/water interface. This conformational ordering may be due to a kinetically limited phase transition taking place in the meniscus formed between the solid substrate and aqueous subphase. In addition, the results obtained for both the phospholipid and fatty acid suggest that the structure of the amphiphile may help determine the extent and nature of the transfer-speed-induced structural changes taking place in the monomolecular film.

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New insights into the structural basis of integrin activation

Blood ◽

10.1182/blood-2003-01-0334 ◽

2003 ◽

Vol 102 (4) ◽

pp. 1155-1159 ◽

Cited By ~ 133

Author(s):

Jian-Ping Xiong ◽

Thilo Stehle ◽

Simon L. Goodman ◽

M. Amin Arnaout

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Structural Basis ◽

Integrin Activation ◽

Cellular Functions ◽

Electron Microscopic ◽

The Past ◽

Intracellular Signals ◽

Bidirectional Signaling ◽

New Hypothesis

Abstract Integrins are cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane and thus influence most cellular functions. Intracellular signals switch integrins into a ligand-competent state as a result of elicited conformational changes in the integrin ectodomain. Binding of extracellular ligands induces, in turn, structural changes that convey distinct signals to the cell interior. The structural basis of this bidirectional signaling has been the focus of intensive study for the past 3 decades. In this perspective, we develop a new hypothesis for integrin activation based on recent crystallographic, electron microscopic, and biochemical studies.

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Metastable Intermediates as Stepping Stones on the Maturation Pathways of Viral Capsids

mBio ◽

10.1128/mbio.02067-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 10

Author(s):

Giovanni Cardone ◽

Robert L. Duda ◽

Naiqian Cheng ◽

Lili You ◽

James F. Conway ◽

...

Keyword(s):

Conformational Changes ◽

Large Scale ◽

Structural Changes ◽

Energy Landscape ◽

Potential Hazard ◽

Stepping Stones ◽

Scanning Calorimetry ◽

Conformational Effects ◽

Capsid Maturation ◽

Capsid Integrity

ABSTRACT As they mature, many capsids undergo massive conformational changes that transform their stability, reactivity, and capacity for DNA. In some cases, maturation proceeds via one or more intermediate states. These structures represent local minima in a rich energy landscape that combines contributions from subunit folding, association of subunits into capsomers, and intercapsomer interactions. We have used scanning calorimetry and cryo-electron microscopy to explore the range of capsid conformations accessible to bacteriophage HK97. To separate conformational effects from those associated with covalent cross-linking (a stabilization mechanism of HK97), a cross-link-incompetent mutant was used. The mature capsid Head I undergoes an endothermic phase transition at 60°C in which it shrinks by 7%, primarily through changes in its hexamer conformation. The transition is reversible, with a half-life of ~3 min; however, >50% of reverted capsids are severely distorted or ruptured. This observation implies that such damage is a potential hazard of large-scale structural changes such as those involved in maturation. Assuming that the risk is lower for smaller changes, this suggests a rationalization for the existence of metastable intermediates: that they serve as stepping stones that preserve capsid integrity as it switches between the radically different conformations of its precursor and mature states. IMPORTANCE Large-scale conformational changes are widespread in virus maturation and infection processes. These changes are accompanied by the release of conformational free energy as the virion (or fusogenic glycoprotein) switches from a precursor state to its mature state. Each state corresponds to a local minimum in an energy landscape. The conformational changes in capsid maturation are so radical that the question arises of how maturing capsids avoid being torn apart. Offering proof of principle, severe damage is inflicted when a bacteriophage HK97 capsid reverts from the (nonphysiological) state that it enters when heated past 60°C. We suggest that capsid proteins have been selected in part by the criterion of being able to avoid sustaining collateral damage as they mature. One way of achieving this—as with the HK97 capsid—involves breaking the overall transition down into several smaller steps in which the risk of damage is reduced.

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Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

International Journal of Molecular Sciences ◽

10.3390/ijms22062937 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2937

Author(s):

Monika Halat ◽

Magdalena Klimek-Chodacka ◽

Jagoda Orleanska ◽

Malgorzata Baranska ◽

Rafal Baranski

Keyword(s):

Circular Dichroism ◽

Conformational Changes ◽

Structural Changes ◽

Electronic Circular Dichroism ◽

Guide Rna ◽

Cas9 Protein ◽

Rnp Complex ◽

Characteristic Features ◽

Circular Dichroïsm

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

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Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

Journal of Virology ◽

10.1128/jvi.78.6.2994-3002.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2994-3002 ◽

Cited By ~ 157

Author(s):

Anne Op De Beeck ◽

Cécile Voisset ◽

Birke Bartosch ◽

Yann Ciczora ◽

Laurence Cocquerel ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Conformational Changes ◽

Structural Changes ◽

Envelope Proteins ◽

Viral Envelope ◽

Envelope Glycoproteins ◽

Functional Envelope

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

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