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NMR assignments of the free and bound-state protein components of an anti-idiotypic affibody complex
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).ORCID-id: 0000-0001-9238-7246
Göteborgs Universitet.
2006 (Engelska)Ingår i: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 36, s. (Electronic publication ahead of print Feb. 6; doi:10.1007/s10858-005-5350-8)Artikel i tidskrift (Refereegranskat) Published
Ort, förlag, år, upplaga, sidor
2006. Vol. 36, s. (Electronic publication ahead of print Feb. 6; doi:10.1007/s10858-005-5350-8)
Nationell ämneskategori
Annan industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-6407DOI: 10.1007/s10858-005-5350-8ISI: 000242836500013Scopus ID: 2-s2.0-33746879057OAI: oai:DiVA.org:kth-6407DiVA, id: diva2:11108
Anmärkning
QC 20101025Tillgänglig från: 2006-11-22 Skapad: 2006-11-22 Senast uppdaterad: 2017-12-14Bibliografiskt granskad
Ingår i avhandling
1. Structural and thermodynamical basis for molecular recognition between engineered binding proteins
Öppna denna publikation i ny flik eller fönster >>Structural and thermodynamical basis for molecular recognition between engineered binding proteins
2006 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA).

In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity.

A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation.

A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2006. s. 57
Nyckelord
protein structure, induced fit, binding thermodynamics, NMR spectroscopy, protein engineering, protein-protein interactions, protein stability, calorimetry
Nationell ämneskategori
Annan industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-4181 (URN)91-7178-481-0 (ISBN)
Disputation
2006-12-01, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Handledare
Anmärkning
QC 20110118Tillgänglig från: 2006-11-22 Skapad: 2006-11-22 Senast uppdaterad: 2011-12-08Bibliografiskt granskad
2. Molecular principles of protein stability and protein-protein interactions
Öppna denna publikation i ny flik eller fönster >>Molecular principles of protein stability and protein-protein interactions
2005 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2005. s. x, 77
Nyckelord
affibody, binding thermodynamics, induced fit, molten globule, NMR spectroscopy, protein engineering, protein-protein interactions, protein stability, protein structure.
Nationell ämneskategori
Annan industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-480 (URN)91-7178-189-7 (ISBN)
Disputation
2005-11-25, E1, Lindstedtvägen 3, Stockholm, 13:00
Opponent
Handledare
Anmärkning
QC 20101025Tillgänglig från: 2005-11-08 Skapad: 2005-11-08 Senast uppdaterad: 2011-12-08Bibliografiskt granskad

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