Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state.

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Structure of Nanobody Nb23

Molecules ◽

10.3390/molecules26123567 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3567

Author(s):

Mathias Percipalle ◽

Yamanappa Hunashal ◽

Jan Steyaert ◽

Federico Fogolari ◽

Gennaro Esposito

Keyword(s):

Nmr Spectroscopy ◽

Chemical Shift ◽

Solution Structure ◽

Chemical Shifts ◽

Molecular Size ◽

Side Chain ◽

3D Nmr ◽

Target Antigen ◽

Internuclear Distances ◽

2D And 3D

Background: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of β2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. Methods: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. Results: The solution structure of isolated Nb23 nanobody was determined. Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

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Backbone NMR assignments of the C-terminal domain of the human prion protein and its disease‐associated T183A variant

Biomolecular NMR Assignments ◽

10.1007/s12104-021-10005-y ◽

2021 ◽

Vol 15 (1) ◽

pp. 193-196

Author(s):

Máximo Sanz-Hernández ◽

Alfonso De Simone

Keyword(s):

Prion Protein ◽

Nmr Assignments ◽

Chemical Shifts ◽

Prion Diseases ◽

Random Coil ◽

Transmissible Spongiform Encephalopathies ◽

Globular Domain ◽

Structural Elements ◽

Spongiform Encephalopathies ◽

Human Prion Protein

AbstractTransmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders associated with the misfolding and aggregation of the human prion protein (huPrP). Despite efforts into investigating the process of huPrP aggregation, the mechanisms triggering its misfolding remain elusive. A number of TSE-associated mutations of huPrP have been identified, but their role at the onset and progression of prion diseases is unclear. Here we report the NMR assignments of the C-terminal globular domain of the wild type huPrP and the pathological mutant T183A. The differences in chemical shifts between the two variants reveal conformational alterations in some structural elements of the mutant, whereas the analyses of secondary shifts and random coil index provide indications on the putative mechanisms of misfolding of T183A huPrP.

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Presence of β-Turn Structure in Recombinant Spider Silk Dissolved in Formic Acid Revealed with NMR

Molecules ◽

10.3390/molecules27020511 ◽

2022 ◽

Vol 27 (2) ◽

pp. 511

Author(s):

Yu Suzuki ◽

Takanori Higashi ◽

Takahiro Yamamoto ◽

Hideyasu Okamura ◽

Takehiro K. Sato ◽

...

Keyword(s):

Mechanical Properties ◽

Formic Acid ◽

Spider Silk ◽

Chemical Shifts ◽

Repetitive Sequences ◽

Silk Protein ◽

Random Coil ◽

Oh Groups ◽

Spider Silk Protein ◽

Turn Structure

Spider dragline silk is a biopolymer with excellent mechanical properties. The development of recombinant spider silk protein (RSP)-based materials with these properties is desirable. Formic acid (FA) is a spinning solvent for regenerated Bombyx mori silk fiber with excellent mechanical properties. To use FA as a spinning solvent for RSP with the sequence of major ampullate spider silk protein from Araneus diadematus, we determined the conformation of RSP in FA using solution NMR to determine the role of FA as a spinning solvent. We assigned 1H, 13C, and 15N chemical shifts to 32-residue repetitive sequences, including polyAla and Gly-rich regions of RSP. Chemical shift evaluation revealed that RSP is in mainly random coil conformation with partially type II β-turn structure in the Gly-Pro-Gly-X motifs of the Gly-rich region in FA, which was confirmed by the 15N NOE data. In addition, formylation at the Ser OH groups occurred in FA. Furthermore, we evaluated the conformation of the as-cast film of RSP dissolved in FA using solid-state NMR and found that β-sheet structure was predominantly formed.

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Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants

Viruses ◽

10.3390/v10090459 ◽

2018 ◽

Vol 10 (9) ◽

pp. 459 ◽

Cited By ~ 3

Author(s):

Wenjie Qiao ◽

Erin Helpio ◽

Bryce Falk

Keyword(s):

Transmembrane Protein ◽

Genomic Analysis ◽

Open Reading Frames ◽

Virus Infectivity ◽

Gene Block ◽

Unfolded Protein ◽

Small Proteins ◽

Lettuce Infectious Yellows Virus ◽

The Unfolded Protein Response ◽

Protein Response

Genomic analysis of Lettuce infectious yellows virus (LIYV) has revealed two short open reading frames (ORFs) on LIYV RNA2, that are predicted to encode a 5-kDa (P5) and a 9-kDa (P9) protein. The P5 ORF is part of the conserved quintuple gene block in the family Closteroviridae, while P9 orthologs are found in all Criniviruses. In this study, the expression of LIYV P5 and P9 proteins was confirmed; P5 is further characterized as an endoplasmic reticulum (ER)-localized integral transmembrane protein and P9 is a soluble protein. The knockout LIYV mutants presented reduced symptom severity and virus accumulation in Nicotiana benthamiana or lettuce plants, indicating their importance in efficient virus infection. The P5 mutant was successfully complemented by a dislocated P5 in the LIYV genome. The structural regions of P5 were tested and all were found to be required for the appropriate functions of P5. In addition, P5, as well as its ortholog P6, encoded by Citrus tristeza virus (CTV) and another ER-localized protein encoded by LIYV RNA1, were found to cause cell death when expressed in N. benthamiana plants from a TMV vector, and induce ER stress and the unfolded protein response (UPR).

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Random coil chemical shifts for serine, threonine and tyrosine phosphorylation over a broad pH range

Journal of Biomolecular NMR ◽

10.1007/s10858-019-00283-z ◽

2019 ◽

Vol 73 (12) ◽

pp. 713-725 ◽

Cited By ~ 4

Author(s):

Ruth Hendus-Altenburger ◽

Catarina B. Fernandes ◽

Katrine Bugge ◽

Micha B. A. Kunze ◽

Wouter Boomsma ◽

...

Keyword(s):

Chemical Shift ◽

Secondary Structure ◽

Intrinsically Disordered Proteins ◽

Chemical Shifts ◽

Random Coil ◽

Disordered Proteins ◽

Phosphoryl Group ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Secondary Chemical

Abstract Phosphorylation is one of the main regulators of cellular signaling typically occurring in flexible parts of folded proteins and in intrinsically disordered regions. It can have distinct effects on the chemical environment as well as on the structural properties near the modification site. Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins (IDPs) and the reliability of the analysis depends on an appropriate choice of random coil model. Random coil chemical shifts and sequence correction factors were previously determined for an Ac-QQXQQ-NH2-peptide series with X being any of the 20 common amino acids. However, a matching dataset on the phosphorylated states has so far only been incompletely determined or determined only at a single pH value. Here we extend the database by the addition of the random coil chemical shifts of the phosphorylated states of serine, threonine and tyrosine measured over a range of pH values covering the pKas of the phosphates and at several temperatures (www.bio.ku.dk/sbinlab/randomcoil). The combined results allow for accurate random coil chemical shift determination of phosphorylated regions at any pH and temperature, minimizing systematic biases of the secondary chemical shifts. Comparison of chemical shifts using random coil sets with and without inclusion of the phosphoryl group, revealed under/over estimations of helicity of up to 33%. The expanded set of random coil values will improve the reliability in detection and quantification of transient secondary structure in phosphorylation-modified IDPs.

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Random coil carbon chemical shifts of deoxyribonucleic acids

Journal of Magnetic Resonance ◽

10.1016/j.jmr.2003.10.001 ◽

2004 ◽

Vol 166 (1) ◽

pp. 11-18 ◽

Cited By ~ 7

Author(s):

Chit Wan Kwok ◽

Cheuk Nang Ho ◽

Lai Man Chi ◽

Sik Lok Lam

Keyword(s):

Chemical Shifts ◽

Random Coil

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Pressure dependence of side chain 1H and 15N-chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2

Journal of Biomolecular NMR ◽

10.1007/s10858-020-00326-w ◽

2020 ◽

Vol 74 (8-9) ◽

pp. 381-399

Author(s):

Markus Beck Erlach ◽

Joerg Koehler ◽

Claudia E. Munte ◽

Werner Kremer ◽

Edson Crusca ◽

...

Keyword(s):

Amino Acids ◽

Pressure Dependence ◽

Chemical Shifts ◽

Model Systems ◽

Random Coil ◽

Nonlinear Dependence ◽

Side Chain ◽

Unfolded Proteins ◽

Pressure Coefficients ◽

Compression Effects

Abstract For interpreting the pressure induced shifts of resonance lines of folded as well as unfolded proteins the availability of data from well-defined model systems is indispensable. Here, we report the pressure dependence of 1H and 15N chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx is one of the 20 canonical amino acids) measured at 800 MHz proton frequency. As observed earlier for other nuclei the chemical shifts of the side chain nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The pressure response is described by a second degree polynomial with the pressure coefficients B1 and B2 that are dependent on the atom type and type of amino acid studied. A number of resonances could be assigned stereospecifically including the 1H and 15N resonances of the guanidine group of arginine. In addition, stereoselectively isotope labeled SAIL amino acids were used to support the stereochemical assignments. The random-coil pressure coefficients are also dependent on the neighbor in the sequence as an analysis of the data shows. For Hα and HN correction factors for different amino acids were derived. In addition, a simple correction of compression effects in thermodynamic analysis of structural transitions in proteins was derived on the basis of random-coil pressure coefficients.

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1H NMR studies on the solution conformation of contulakin-G and analogues

Canadian Journal of Chemistry ◽

10.1139/v02-115 ◽

2002 ◽

Vol 80 (8) ◽

pp. 1022-1031 ◽

Cited By ~ 9

Author(s):

Lill Kindahl ◽

Corine Sandström ◽

A Grey Craig ◽

Thomas Norberg ◽

Lennart Kenne

Keyword(s):

Hydrogen Bond ◽

Chemical Shifts ◽

Torsional Rigidity ◽

Peptide Chain ◽

Random Coil ◽

Amide Proton ◽

Solution Conformation ◽

Nmr Studies ◽

H Nmr ◽

Intramolecular Hydrogen

The conformation of contulakin-G, a bioactive 16 amino acid O-linked glycopeptide (ZSEEGGSNAT*KKPYIL) with the disaccharide β-D-Gal(1[Formula: see text]3)α-D-GalNAc attached to the threonine residue in position 10, has been investigated by 1H NMR spectroscopy. The 1H NMR data for the non-glycosylated peptide and for two glycopeptide analogues, one with the monosaccharide α-D-GalNAc at Thr10 and one with the disaccharide β-D-Gal(1–>3)α-D-GalNAc at Ser7, all of lower bioactivity than contulakin-G, have also been collected. The chemical shifts, NOEs, temperature coefficients of amide protons, and 3JNH,αH-values suggest that all four compounds exist mainly in random coil conformations. Some transient populations of folded conformations are also present in the glycopeptides and turns, probably induced by the sugars, are present in the peptide chain around the site of glycosylation. In the two peptides O-glycosylated at Thr10, the rotation of α-D-GalNAc around the linkage between the sugar and the peptide is restricted. There is evidence for a hydrogen bond between the amide proton of α-D-GalNAc and the peptide chain that could contribute to this torsional rigidity. An intramolecular hydrogen bond between the carbohydrate and the peptide chain does not exist in the peptide O-glycosylated at the Ser7 residue. Key words: conformation, contulakin-G, NMR, O-linked glycopeptide.

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CheSPI: Chemical shift Secondary structure Population Inference

10.1101/2021.02.20.432095 ◽

2021 ◽

Author(s):

Jakob Toudahl Nielsen ◽

Frans A.A. Mulder

Keyword(s):

Intrinsically Disordered Proteins ◽

Chemical Shifts ◽

Protein Structures ◽

Random Coil ◽

Disordered Proteins ◽

Residual Structure ◽

Intrinsically Disordered ◽

Population Inference ◽

Local Protein Structure ◽

Structural Classes

AbstractNMR chemical shifts (CSs) are delicate reporters of local protein structure, and recent advances in random coil CS (RCCS) prediction and interpretation now offer the compelling prospect of inferring small populations of structure from small deviations from RCCSs. Here, we present CheSPI, a simple and efficient method that provides unbiased and sensitive aggregate measures of local structure and disorder. It is demonstrated that CheSPI can predict even very small amounts of residual structure and robustly delineate subtle differences into four structural classes for intrinsically disordered proteins. For structured regions and proteins, CheSPI can assign up to eight structural classes, which coincide with the well-known DSSP classification. The program is freely available, and can either be invoked from URL www.protein-nmr.org as a web implementation, or run locally from command line as a python program. CheSPI generates comprehensive numeric and graphical output for intuitive annotation and visualization of protein structures. A number of examples are provided.

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Electrical unfolding of cytochrome c during translocation through a nanopore constriction

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2016262118 ◽

2021 ◽

Vol 118 (17) ◽

pp. e2016262118

Author(s):

Prabhat Tripathi ◽

Abdelkrim Benabbas ◽

Behzad Mehrafrooz ◽

Hirohito Yamazaki ◽

Aleksei Aksimentiev ◽

...

Keyword(s):

Free Energy ◽

Electric Field ◽

Electric Fields ◽

Driving Force ◽

Protein Translocation ◽

Critical Factors ◽

Unfolded Protein ◽

Small Proteins ◽

Unfolded State ◽

Cellular Compartments

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field–driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field–induced deformability.

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