Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

Goehlert, Virginia A.; Krupinska, Ewa; Regan, Lynne; Stone, Martin J.

doi:10.1110/ps.04926604

Cited by 14 publications

(7 citation statements)

References 39 publications

(54 reference statements)

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“…The chemical shift differences were not simply a result of the chemical differences between the model system and the full-length protein in its amyloid form. Characterization of a variety of mutated forms of the B1 domain of protein G revealed that the average changes in chemical shift from a nonstructurally perturbative mutation are small (∼0.1 ppm in 13 C and 0.3 ppm in 15 N) (40). The one-bond TEDOR spectrum of a uniformly 13 C-and 15 N-labeled NM fiber sample at room temperature (A) is greatly simplified by using an NM molecule that is isotopically labeled at only the first 14 amino acids (B) (red).…”

Section: Resultsmentioning

confidence: 99%

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Frederick

Michaelis

Caporini

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C-and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. (2), is an amyloid form of the translation termination factor Sup35 (3). The amyloid fold is polymeric fiber of protein monomers that is rich in β-sheets that run perpendicular to the fiber axis. Amyloid formation sequesters Sup35 from the ribosome, changing the rate of stop-codon read-through and creating a variety of new yeast phenotypes (4,5). Different regions of the Sup35 protein are responsible for prion templating, prion inheritance, and translation termination. The N-terminal domain "N" is Q/N-rich and required for the formation and templating of amyloid. The highly charged K/E-rich middle domain "M" promotes solubility in the nonprion form and is required for chaperone-mediated prion inheritance (6, 7). The C-terminal domain is necessary and sufficient for translation termination. The N and M domains together, NM, contain all of the necessary features to act as an amyloid-based prion.A wide variety of approaches have been used to investigate Sup35 prion structures, but a consensus structural picture has yet to emerge. Amyloids are notoriously difficult to study, mainly because they are insoluble yet noncrystalline and are therefore not easily amenable to traditional structural biology methods. In all models, at least some part of the N domain is involved in the amyloid fold (8-11), and most of the M domain is thought to be solvent-accessible and intrinsically disordered (11-13). However, there are two conflicting models of how monomers of NM are arranged in amyloid fibers. In one model, called the β-helical model, monomers make intermolecular contacts in discrete regions, with the region in-between making intramolecular contacts. This model is supported by the patterns of interaction and cross-linking determined from a series of site-directed mutants (8). The work has the caveat that mutations may perturb the fibril structure. However, fibers made from the mutated versions of the protein ca...

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Section: Resultsmentioning

confidence: 99%

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Frederick

Michaelis

Caporini

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…In a “typical” analysis, values of τ f and are extracted from fits of R 1 , R 2 rates to the model described above, eqs 1–3, assuming a value for τ C that is obtained from backbone 15 N spin relaxation experiments [36], [47]–[49]. Here we have taken a different strategy that avoids complications that emerge from a potentially erroneous τ C value resulting from 15 N relaxation rates “contaminated” by chemical exchange.…”

Section: Resultsmentioning

confidence: 99%

Is Buffer a Good Proxy for a Crowded Cell-Like Environment? A Comparative NMR Study of Calmodulin Side-Chain Dynamics in Buffer and E. coli Lysate

Latham

Kay

2012

PLoS ONE

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Biophysical studies of protein structure and dynamics are typically performed in a highly controlled manner involving only the protein(s) of interest. Comparatively fewer such studies have been carried out in the context of a cellular environment that typically involves many biomolecules, ions and metabolites. Recently, solution NMR spectroscopy, focusing primarily on backbone amide groups as reporters, has emerged as a powerful technique for investigating protein structure and dynamics in vivo and in crowded “cell-like” environments. Here we extend these studies through a comparative analysis of Ile, Leu, Val and Met methyl side-chain motions in apo, Ca2+-bound and Ca2+, peptide-bound calmodulin dissolved in aqueous buffer or in E. coli lysate. Deuterium spin relaxation experiments, sensitive to pico- to nano-second time-scale processes and Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, reporting on millisecond dynamics, have been recorded. Both similarities and differences in motional properties are noted for calmodulin dissolved in buffer or in lysate. These results emphasize that while significant insights can be obtained through detailed “test-tube” studies, experiments performed under conditions that are “cell-like” are critical for obtaining a comprehensive understanding of protein motion in vivo and therefore for elucidating the relation between motion and function.

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“…The proteins used in this study are: ubiquitin (PDB id: 1UBQ) 43 , the third fibronectin type III domain from human tenascin (TNfn3; PDB id: 1TEN) 44 , the tenth fibronectin type III domain from human fibronectin (FNfn10; PDB id: 1FNA) 45 , the B1 domain from protein G (gb1; PDB id: 1PGB) 46 , the SH3 domain in human Fyn (fyn-sh3; PDB id: 1SHF) 34 , the de novo designed three-helix bundle α3D (a3D; PDB id: 2A3D) 6 , eglin c (PDB id: 1CSE) 7; 47 , an SH2 domain of phospholipase C-gamma1 (plcc-sh2; PDB id: 2PLD) 7 , the B1 domain of protein L (proteinL; PDB id: 1HZ6) 31 , HIV-1 protease homodimer bound to the inhibitor DMP323, (hiv-protease-holo; PDB id: 1QBS), flavodoxin bound to flavin mononucleotide (flavodoxin-holo; PDB id: 1OBO) 48 , cytochrome c2 bound to its heme prosthetic group (cytochrome-c2-holo; PDB id: 1C2R) 49 , the N-terminal domain of chicken skeletal troponin C (NTnC; PDB id: 1AVS) 50 , Cdc42Hs 51 , adipocyte lipid-binding protein (albp; PDB id: 1LIB) 52 , muscle fatty acid-binding protein (mfabp; PDB id: 1HMT) 52 , and Ca 2+ -loaded calmodulin (PDB id: 1AHR) 53 .…”

Section: Dataset Of Experimental Protein Structures and Relaxation Mementioning

confidence: 99%

A Simple Model of Backbone Flexibility Improves Modeling of Side-chain Conformational Variability

Friedland

Linares

Smith

et al. 2008

Journal of Molecular Biology

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The considerable flexibility of side-chains in folded proteins is important for protein stability and function, and may play a role in mediating pathways of energetic connectivity between allosteric sites. While sampling side-chain degrees of freedom has been an integral part of several successful computational protein design methods, the predictions of these approaches have not been directly compared to experimental measurements of side-chain motional amplitudes. In addition, protein design methods generally keep the backbone fixed, an approximation that may substantially limit the ability to accurately model side-chain flexibility. Here we describe a Monte Carlo approach to modeling side-chain conformational variability and validate our method against a large dataset of methyl relaxation order parameters derived from Nuclear Magnetic Resonance experiments (17 proteins and a total of 530 data points). We also evaluate a model of backbone flexibility based on Backrub motions, a type of conformational change frequently observed in ultra-high resolution Xray structures that accounts for correlated side-chain backbone movements. The fixed-backbone model performs reasonably well with an overall rmsd between computed and predicted side-chain order parameters of 0.26. Notably, including backbone flexibility leads to significant © 2008 Elsevier Ltd. All rights reserved. * Corresponding author: Kortemme@cgl.ucsf.edu. AUTHOR CONTRIBUTIONSGDF and TK conceived and designed the experiments. GDF and AJL performed the experiments. GDF, TK, and AJL analyzed the data and wrote the paper. CAS contributed reagents/materials/analysis tools. SUPPORTING INFORMATION AVAILABLEOne figure with population distributions of ubiquitin I13 and L15 chi 1 and chi 2. One table with detailed results from Model 1; one table with results from Models 2 and 3 for nonpolar methyl groups only; and one table with detailed parameter sensitivity results from Model 2.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

Cited by 14 publications

References 39 publications

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Is Buffer a Good Proxy for a Crowded Cell-Like Environment? A Comparative NMR Study of Calmodulin Side-Chain Dynamics in Buffer and E. coli Lysate

A Simple Model of Backbone Flexibility Improves Modeling of Side-chain Conformational Variability

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