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Structure determination and thermodynamic stabilization of an engineered protein-protein complex
KTH, School of Biotechnology (BIO).
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity.

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , vii, 67 p.
Keyword [en]
affibody, protein structure, coupled folding, NMR spectroscopy, protein stability, protein-protein interactions, binding thermodynamics, isothermal titration calorimetry, protein engineering
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4230ISBN: 91-7178-457-8 (print)OAI: oai:DiVA.org:kth-4230DiVA: diva2:11367
Public defence
2006-12-15, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:30
Opponent
Supervisors
Note
QC 20100924Available from: 2006-12-12 Created: 2006-12-12 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
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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.
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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
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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. Conformational Stabilization of an Engineered Binding Protein
Open this publication in new window or tab >>Conformational Stabilization of an Engineered Binding Protein
2006 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 23, 7651-7660 p.Article in journal (Refereed) Published
Abstract [en]

We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA-1) affibody binds to its Z domain binding partner with a dissociation constant K-d = 1.6 mu M, and previous analyses suggested that the moderate \affinity is due to the conformational heterogeneity of free Z(SPA-1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA-1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K-d = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA-1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA-1). Instead, higher Z domain binding affinity in a group of eight Z(SPA-1) variants correlates with more favorable binding enthalpy and enthalpy- entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.

Keyword
disulfide bonds, combinatorial libraries, molten globules, stability, domain, affibody, complex, thermodynamics, hydration, lysozyme
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6611 (URN)10.1021/ja060933g (DOI)000238099500055 ()2-s2.0-33745100912 (Scopus ID)
Note
QC 20100924Available from: 2006-12-12 Created: 2006-12-12 Last updated: 2010-09-24Bibliographically approved

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