Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • 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
Molecular principles of protein stability and protein-protein interactions
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-9238-7246
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.

Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , x, 77 p.
Keyword [en]
affibody, binding thermodynamics, induced fit, molten globule, NMR spectroscopy, protein engineering, protein-protein interactions, protein stability, protein structure.
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-480ISBN: 91-7178-189-7 (print)OAI: oai:DiVA.org:kth-480DiVA: diva2:14025
Public defence
2005-11-25, E1, Lindstedtvägen 3, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20101025Available from: 2005-11-08 Created: 2005-11-08 Last updated: 2011-12-08Bibliographically approved
List of papers
1. 1H, 13C and 15N resonance assignments of an affibody-target complex
Open this publication in new window or tab >>1H, 13C and 15N resonance assignments of an affibody-target complex
Show others...
2002 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 24, no 3, 271-272 p.Article in journal (Refereed) Published
Keyword
Affibody, Molecular recognition, NMR assignment, Protein A, Protein engineering, Protein-Protein interactions, Z domain
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8641 (URN)10.1023/A:1021664710731 (DOI)
Note
QC 20100924Available from: 2005-11-08 Created: 2005-11-08 Last updated: 2010-09-24Bibliographically approved
2. An affibody in complex with a target protein: Structure and coupled folding.
Open this publication in new window or tab >>An affibody in complex with a target protein: Structure and coupled folding.
Show others...
2003 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 6, 3185-3190 p.Article in journal (Refereed) Published
Abstract [en]

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z(SPA-1) affibody has been selected from a phage-displayed library as a binder to protein A. Z(SPA-1) also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z(SPA-1) affibody in its uncomplexed state and determined the solution structure of a Z:Z(SPA-1) protein-protein complex. Uncomplexed Z(SPA-1) behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z(SPA-1). We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z(SPA-1) and Ig Fc, respectively, suggesting that the Z(SPA-1) affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.

Keyword
protein engineering, protein-protein interactions, molecular recognition, NMR spectroscopy, induced fit
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8642 (URN)10.1073/pnas.0436086100 (DOI)000181675200039 ()
Note
Uppdaterad från "In press" till published: 20100924. QC 20100924Available from: 2005-11-08 Created: 2005-11-08 Last updated: 2010-09-24Bibliographically approved
3. Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A
Open this publication in new window or tab >>Biophysical characterization of ZSPA-1-A phage-display selected binder to protein A
Show others...
2004 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, no 8, 2078-2088 p.Article in journal (Refereed) Published
Abstract [en]

Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

Keyword
protein engineering, affibody, protein stability, osmolyte, NMR spectroscopy
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8643 (URN)10.1110/ps.04728604 (DOI)000222928800010 ()2-s2.0-3342879396 (Scopus ID)
Note

QC 20100924

Available from: 2005-11-08 Created: 2005-11-08 Last updated: 2017-06-14Bibliographically approved
4. Thermodynamics of folding, stabilization, and binding in an engineered protein-protein complex
Open this publication in new window or tab >>Thermodynamics of folding, stabilization, and binding in an engineered protein-protein complex
Show others...
2004 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 126, no 36, 11220-11230 p.Article in journal (Refereed) Published
Abstract [en]

We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA-1) affibody and it's Z domain binding partner as a model. Free Z(SPA-1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA-1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA-1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA-1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA-1) complex is associated with a large change in conformational entropy that opposes binding.

Keyword
combinatorial libraries, staphylococcus-aureus, molten globules, affibody, domain, entropy, dna, microcalorimetry, recognition, parameters
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6406 (URN)10.1021/ja047727y (DOI)000223799900035 ()2-s2.0-4544277195 (Scopus ID)
Note
QC 20101025 QC 20110916Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2011-09-16Bibliographically approved
5. NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex
Open this publication in new window or tab >>NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex
2006 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 36, (Electronic publication ahead of print Feb. 6; doi:10.1007/s10858-005-5350-8) p.Article in journal (Refereed) Published
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6407 (URN)10.1007/s10858-005-5350-8 (DOI)000242836500013 ()2-s2.0-33746879057 (Scopus ID)
Note
QC 20101025Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2010-10-25Bibliographically approved
6. Structural basis for molecular recognition in an affibody:affibody complex
Open this publication in new window or tab >>Structural basis for molecular recognition in an affibody:affibody complex
2006 (English)In: Asia-Pacific Journal of Molecular Biology and Biotechnology, ISSN 0128-7451, Vol. 359, no 5, 1293-1304 p.Article in journal (Refereed) Published
Abstract [en]

Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K-d similar to 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large (similar to 1670 angstrom(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine H alpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium (NH2)-H-eta groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.

Keyword
protein-protein interactions, protein engineering, molecular recognition, NMR spectroscopy, induced fit
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6408 (URN)10.1016/j.jmb.2006.04.043 (DOI)000238988400011 ()2-s2.0-33746933474 (Scopus ID)
Note
QC 20101025Available from: 2006-11-22 Created: 2006-11-22 Last updated: 2010-10-25Bibliographically approved

Open Access in DiVA

fulltext(12585 kB)1046 downloads
File information
File name FULLTEXT01.pdfFile size 12585 kBChecksum MD5
8ecb1f74a0735fe10b3e238ef02d3c0a99ee1ede0d500d690d8392da3880c9913a68a49e
Type fulltextMimetype application/pdf

Authority records BETA

Lendel, Christofer

Search in DiVA

By author/editor
Lendel, Christofer
By organisation
School of Biotechnology (BIO)
Other Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1046 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: 1287 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • 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