Systematic mapping of free energy landscapes of a growing filamin domain during biosynthesis

Waudby, Christopher A.; Włodarski, Tomasz; Karyadi, Maria-Evangelia; Cassaignau, Anaïs M E; Chan, Sammy H. S.; Wentink, Anne S.; Schmidt-Engler, Julian M.; Camilloni, Carlo; Vendruscolo, Michele; Cabrita, Lisa D.; Christodoulou, John

doi:10.1073/pnas.1716252115

Cited by 44 publications

(80 citation statements)

References 29 publications

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“…3, together with a curve derived from the NMR data showing averaged relative intensities of three 15 N amide resonances arising from the unfolded FLN5 domain [14]. To be consistent with the published NMR data [13,14], in this case we plot the data as a function of L * , i.e., the distance between the C-terminal end of the FLN5 domain and the PTC (c.f., Fig. 1b).…”

Section: Cotranslational Folding Of the Fln5 Domainmentioning

confidence: 94%

“…The cotranslational folding of the Ig-like FLN5 filamin domain has previously been studied by NMR measurements on ribosome-attached nascent chains and of purified C-terminally truncated versions of the protein [13,14]. The main conclusion from these studies is that FLN5 folds only when it has fully cleared the exit tunnel and is some distance away from the ribosome surface, at a tether length of 40-45 residues.…”

Section: Cotranslational Folding Of the Fln5 Domainmentioning

confidence: 99%

“…In recent years, a number of new experimental methods for analyzing the cotranslational folding of protein domains have been developed. These include real-time FRET analysis [1], and methods in which nascent polypeptide chains of defined lengths are arrested in the ribosome and their folding status analyzed by, e.g., cryo-EM [2,3], protease resistance [1,[4][5][6][7][8][9], NMR [10][11][12][13][14][15], photoinduced electron transfer (PET) [1,16], folding-associated cotranslational sequencing [17], optical tweezer pulling [18][19][20][21], fluorescence measurements [22], and measuring the force that the folding protein exerts on the nascent chain using a translational arrest peptide (AP) as a force sensor [2,3,9,21,[23][24][25]. Further, coarse-grained molecular dynamics simulations of various flavors can provide detailed insights into cotranslational folding reactions [26][27][28][29][30], especially when coupled with experimental studies [3,26,31].…”

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confidence: 99%

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Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

Kemp

Kudva

Rosa

et al. 2018

Preprint

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We have characterized the cotranslational folding of two small protein domains of different folds -the α-helical N-terminal domain of HemK and the β-rich FLN5 filamin domain -by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (Force-Profile Analysis -FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: realtime FRET, PET, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.

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Section: Cotranslational Folding Of the Fln5 Domainmentioning

confidence: 94%

Section: Cotranslational Folding Of the Fln5 Domainmentioning

confidence: 99%

mentioning

confidence: 99%

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Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

Kemp

Kudva

Rosa

et al. 2018

Preprint

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“…We have validated our new experiment, which we term the SOFAST-H(Z/D)QC, by comparison with 1 H, 15 N SOFAST-HMQC measurements of the folded protein ubiquitin, and the unfolded Y719E variant of the FLN5 filamin domain 36,37 . Analysis of the integrated amide signal intensity as a function of the inter-scan delay showed that for both samples the sensitivity of SOFAST-HZQC and SOFAST-HDQC experiments was very close to the expected factor of √ 2 less than the SOFAST-HMQC (Fig.…”

Section: /19mentioning

confidence: 99%

Two-dimensional NMR lineshape analysis of single, multiple, zero and double quantum correlation experiments

Waudby

Ouvry

Davis³

et al. 2019

Preprint

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NMR spectroscopy provides a powerful approach for the characterisation of chemical exchange and molecular interactions by analysis of series of experiments acquired over the course of a titration measurement. The appearance of NMR resonances undergoing chemical exchange depends on the frequency difference relative to the rate of exchange, and in the case of one-dimensional experiments chemical exchange regimes are well established and well known. However, two-dimensional experiments present additional complexity, as at least one additional frequency difference must be considered. Here we provide a systematic classification of chemical exchange regimes in two-dimensional NMR spectra. We highlight important differences between exchange in HSQC and HMQC experiments, that on a practical level result in more severe exchange broadening in HMQC spectra, but show that complementary alternatives to the HMQC are available in the form of HZQC and HDQC experiments. We present the longitudinal relaxation optimised SOFAST-H(Z/D)QC experiment for the simultaneous acquisition of sensitivity-enhanced HZQC and HDQC spectra, and the longitudinal and transverse relaxation optimised BEST-ZQ-TROSY for analysis of large molecular weight systems. We describe the application of these experiments to the characterisation of the interaction between the Hsp90 N-terminal domain and a small molecule ligand, and show that the independent analysis of HSQC, HMQC, HZQC and HDQC experiments provides improved confidence in the fitted dissociation constant and dissociation rate. Joint analysis of such data may provide improved sensitivity to detect and analyse more complex multi-state interaction mechanisms such as induced fit or conformational selection.

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“…It is critical, therefore, that this initial folding event proceeds with fidelity and avoids toxic misfolded states (1,2). Both the vectorial nature of translation and interactions with the ribosome can affect this process (3)(4)(5)(6)(7)(8), such that the co-translational folding pathway can be different from the pathway observed during re-folding experiments. For instance, co-translational folding of firefly luciferase promotes formation of an intermediate that helps to prevent misfolding (9).…”

Section: Introductionmentioning

confidence: 99%

The folding and unfolding behavior of ribonuclease H on the ribosome

Jensen

Samelson

Steward

et al. 2020

Preprint

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Folding and unfolding behavior of RNase H on the ribosome 2 ABSTRACT The health of a cell depends on accurate translation and proper protein folding; misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the energy landscape of the nascent chain. However, quantifying these environmental effects is challenging due to the ribosomal proteins and rRNA, which preclude most spectroscopic measurements of protein energetics. We have applied two gel-based approaches, pulse proteolysis and force-peptide arrest assays, to probe the folding and unfolding pathways of RNase H ribosome-stalled nascent chains. We find that ribosome-stalled RNase H has an increased unfolding rate compared to free RNase H, which completely accounts for observed changes in protein stability and indicates that the folding rate is unchanged. Using arrest peptide-based force-profile analysis, we assayed the force generated during the folding of RNase H on the ribosome. Surprisingly, we find that population of the RNase H folding intermediate is required to generate sufficient force to release the SecM stall and that readthrough of the stall sequence directly correlates with the stability of the folding intermediate. Together, these data imply that the folding pathway of RNase H is unchanged on the ribosome. Furthermore, our data indicate that the ribosome promotes unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of misfolding and assist in folding fidelity.An alternative technique to measure folding kinetics and energetics in a complex mixture is pulse proteolysis. Under appropriate conditions, a short pulse of proteolysis degrades unfolded proteins, leaving the population of folded proteins intact. Unlike limited proteolysis, which reports on the relative proteolytic

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Systematic mapping of free energy landscapes of a growing filamin domain during biosynthesis

Cited by 44 publications

References 29 publications

Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

Two-dimensional NMR lineshape analysis of single, multiple, zero and double quantum correlation experiments

The folding and unfolding behavior of ribonuclease H on the ribosome

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