Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Effects of Macromolecular Crowding on Protein Folding: - in-vitro equilibrium and kinetic studies on selected model systems
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Pernilla Wittungs-Stafshede)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein folding is the process during which an extended and unstructured polypeptide converts to its compact folded structure that is most often the functional state. The process has been characterized extensively in dilute buffer in-vitro during the last decades but the actual biological place for this process is the inside of living cells. The cytoplasm of a cell is filled with a plethora of different macromolecules that together occupy up to 40% of the total volume. This large amount of macromolecules restricts the available space to each individual molecule, which has been termed macromolecular crowding. Macromolecular crowding results in excluded volume effects and also increases chances for non-specific interactions. Macromolecular crowding should favor reactions that lead to a decrease in the total occupied volume by all molecules, such as folding reactions. Theoretical models have predicted that the stability of protein folded states should increase in presence of macromolecular crowding due to unfavorable effects on the extended unfolded state. To understand protein folding and function in living systems, we need to have a defined quantitative link between in-vitro dilute conditions (where most biophysical experiments are made) and in-vivo crowded conditions. An important question is thus how macromolecular crowding modifies the biophysical properties of a protein.

The work underlying this thesis focused on how macromolecular crowding tunes protein equilibrium stability and kinetic folding processes. To mimic the crowded cellular environment, synthetic sugar-based polymers (Dextrans of different sizes and Ficoll 70) were used as crowding agents (crowders) in controlled in-vitro experiments. In contrast to previous studies which often have focused on one protein and one crowder at a time, the goal here was to make systematic analyses of how size, shape and concentration of the crowders affect both equilibrium and kinetic properties of structurally-different proteins. Three model proteins (cytochrome c, apoazurin and apoflavodoxin) were investigated under crowding by Ficoll 70 and different-size Dextrans, using various spectroscopic techniques such as far-UV circular dichroism and intrinsic tryptophan fluorescence. Thermodynamic models were applied to explain the experimental results.

It was discovered that equilibrium stability of all three proteins increased in presence of crowding agents in a crowder concentration dependent manner. The stabilization effect was around 2-3 kJ/mol, larger for the various Dextrans than for Ficoll 70 at the same g/l, but independent of Dextran size (in the range 20 to 70 kDa). To further investigate the cause for the stabilization a theoretical crowding model was applied. In this model, Dextran and Ficoll were modeled as elongated rods and the protein was represented as a sphere, where the folded sphere representation was smaller than the unfolded sphere representation. It is notable that the observed stability changes could be reproduced by this model taking only steric interactions into account. This correlation showed that when using sugar-based crowding agents, excluded volume effects could be studied in isolation and there were no contributions from nonspecific interactions.

Time-resolved experiments with apoazurin and apoflavodoxin revealed an increase in the folding rate constants while the unfolding rates were invariant in the presence of crowding agents. For apoflavodoxin and cytochrome c, the presence of crowding agents also altered the folding pathway such that it became more homogeneous (cytochrome c) and it gave less misfolding (apoflavodoxin). These results showed that macromolecular crowding restricts the conformational space of the unfolded polypeptide chain, makes the conformations more compact which, in turn, eliminates access to certain pathways.

The results from kinetic and equilibrium measurements on three model proteins, together with available data from the literature, demonstrate that macromolecular crowding effects due to volume exclusion are in the order of a few kJ/mol. Considering the numerous concentration balances and cross-dependent reactions of the cellular machinery, small changes in energetics/kinetics of the magnitudes found here can still have dramatic consequences for cellular fitness. In fact local and transient changes in macromolecular crowding levels may be a way to tune biochemical reactions without invoking gene expression. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2013. , 85 p.
Keyword [en]
Protein Folding, macromolecular crowding, excluded volume, spectroscopy, stopped-flow
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-82059ISBN: 978-91-7459-764-6 (print)OAI: oai:DiVA.org:umu-82059DiVA: diva2:665133
Public defence
2013-12-11, KBC-Huset, KB3B1, Umeå universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2013-11-20 Created: 2013-10-26 Last updated: 2013-11-19Bibliographically approved
List of papers
1. Factors defining effects of Macromolecular crowding on protein stability: an in vitro/in silico case study using cytochrome c
Open this publication in new window or tab >>Factors defining effects of Macromolecular crowding on protein stability: an in vitro/in silico case study using cytochrome c
Show others...
2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 31, 6519-6530 p.Article in journal (Refereed) Published
Abstract [en]

Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2010
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-35248 (URN)10.1021/bi100578x (DOI)000280743200003 ()20593812 (PubMedID)
Available from: 2010-08-11 Created: 2010-08-11 Last updated: 2017-12-12Bibliographically approved
2. Quantification of excluded volume effects on the folding landscape of Pseudomonas aeruginosa Apoazurin In Vitro
Open this publication in new window or tab >>Quantification of excluded volume effects on the folding landscape of Pseudomonas aeruginosa Apoazurin In Vitro
2013 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 105, no 7, 1689-1699 p.Article in journal (Refereed) Published
Abstract [en]

Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.

Place, publisher, year, edition, pages
Elsevier, 2013
National Category
Biophysics
Research subject
biological chemistry; Biochemistry
Identifiers
urn:nbn:se:umu:diva-82058 (URN)10.1016/j.bpj.2013.08.038 (DOI)24094410 (PubMedID)
Available from: 2013-10-26 Created: 2013-10-26 Last updated: 2017-12-06Bibliographically approved
3. Macromolecular crowding tunes folding landscape of parallel α/β protein, apoflavodoxin
Open this publication in new window or tab >>Macromolecular crowding tunes folding landscape of parallel α/β protein, apoflavodoxin
2011 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 4, 646-648 p.Article in journal (Refereed) Published
Abstract [en]

Proteins normally fold in crowded cellular environments. Here we use a set of Desulfovibrio desulfuricans apoflavodoxin variants to assess-with residue-specific resolution-how apoflavodoxin's folding landscape is tuned by macromolecular crowding. We find that, under crowded conditions, initial topological frustration is reduced, subsequent folding requires less ordering in the transition state, and β-strand 1 becomes more important in guiding the process. We propose that conditions more closely mimicking the cellular environment make the ensemble of unfolded conformations less expanded, resulting in a folding funnel that is smoother and narrower.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2011
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-38759 (URN)10.1021/ja107638e (DOI)21175168 (PubMedID)
Available from: 2010-12-28 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved
4. Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding
Open this publication in new window or tab >>Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding
Show others...
2012 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 49, 9836-9845 p.Article in journal (Refereed) Published
Abstract [en]

Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-64042 (URN)10.1021/bi301324y (DOI)000312122700009 ()
Available from: 2013-01-22 Created: 2013-01-14 Last updated: 2017-12-06Bibliographically approved

Open Access in DiVA

Effects of Macromolecular Crowding on Protein Folding(2843 kB)1033 downloads
File information
File name FULLTEXT01.pdfFile size 2843 kBChecksum SHA-512
c788c4fabaabe057cd76a61edf38f5150776a15b3177c909199e1b1347c2e532bf6b8f6b9d08e0141074c0b6a59112155b03e447c02ff5e00e1beb80670e70a3
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Christiansen, Alexander
By organisation
Department of Chemistry
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 1033 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 442 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf