Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition. Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations? Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle. Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone. Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.

<p>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 Z_SPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: Z_Taq, originally selected to bind Taq DNA polymerase, and anti-Z_Taq, an anti-idiotypic binder to Z_Taq 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 Z_SPA-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 Ų total surface area and 10 out of 13 varied residues in Z_SPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of Z_SPA-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 Z_SPA-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 Z_SPA-1 upon binding seriously reduces the binding affinity. The Z_Taq:anti-Z_Taq complex buries in total 1672 Ų surface area and all varied positions in anti-Z_Taq are directly involved in binding. The main differences between the Z:Z_SPA-1 and the Z_Taq:anti-Z_Taq 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 Z_Taq nor anti-Z_Taq shows the molten globule behaviour seen for Z_SPA-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.</p>

Un polypeptide néosynthétisé est capable de trouver rapidement le chemin vers sa structure tri-dimensionnelle finale, en passant par des intermédiaires partiellement structurés. L'acquisition d'information sur le rôle et l'importance de ces intermédiaires est rendue difficile parce qu'ils se forment très rapidement pendant la réaction de repliement et que cette période de temps n'est pas accessible aux appareils de mélange usuels.<br />L'objet de cette thèse était de caractériser les évènements cinétiques initiaux du repliement des protéines, notamment de l'apomyoglobine (apoMb), en utilisant des appareils de mélange ultra-rapide. Un appareil de type stopped-flow équipé d'une micro-cuve a permis de diminuer le temps mort de ce type de mélangeur. Une réaction bimoléculaire (NATA et NBS), à permis d'évaluer le temps mort à 400±10 µs, dans un mode d'utilisation permettant de suivre simultanément le signal de fluorescence et le signal de dichroïsme circulaire dans l'UV lointain. L'apoMb est une protéine particulièrement intéressante pour l'étude des évènements précoces du repliement des protéines. Le stopped-flow ultra rapide, a permis de suivre des cinétiques (k jusqu'à 1500 s-1) et montré que chaque étape précédemment identifiée, conduisant l'apoMb de sa forme dépliée à sa forme native (soit les réactions UIa, IaIb, et IbN), présente les caractéristiques typiques d'une réaction à deux états, hautement coopérative.<br />Nous avons étudié l'effet d'osmolytes sur les cinétiques et sur la stabilité à l'équilibre des formes U, I et N de l'apoMb. Des études cinétiques en présence de sucrose ont permis d'observer le comportement de la réactions UIa. Ces résultats indiquent que le sucrose déstabilise de manière relative la forme U et l'état de transition de la réaction UIa, par rapport à la forme Ia. L'étape limitante ne correspondrait donc pas à une compaction de la chaîne peptidique. Dans les mêmes conditions, l'étude de la transition IbN permet d'observer que l'état de transition présente des caractéristiques proches de Ib. Ces résultats, décrivant l'effet osmophobique sur l'intermédiaire I, ainsi que des résultats préliminaires de l'effet d'encombrement moléculaire sur le repliement du cytochrome C, sont discutés dans ce mémoire.

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. / QC 20101025

The human &alpha;-lactalbumin (&alpha;-LA) molten globule formed at low pH is a model for the study of protein folding intermediates. The molten globule lacks native-like side-chain interactions, resulting in a fluctuating ensemble of tertiary structures, characterisation of which has been precluded by severe line-broadening in NMR spectra and a lack of long-range NOEs. Paramagnetic relaxation enhancements (PREs) have been measured in a variant of &alpha;-LA in which all native cysteines have been mutated to alanine (all-Ala &alpha;-LA). Cysteine residues have been mutated into regions of interest and spin labelled with MTSL. These measurements have confirmed that all-Ala &alpha;-LA forms a compact molten globule. Transient, long-range interactions that are stabilising the compact fold have also been identified using PREs measured in urea-denatured states. This has identified several interactions formed by hydrophobic residues from both the &alpha;- and &beta;-domain, which could be important for initiating and driving folding. The molten globule’s 3D topology has been probed by measuring long-range distances between MTSL pairs using Double Electron-Electron Resonance (DEER). Broad distance distributions have been identified between elements of secondary structure, indicative of a fluctuating but compact fold. By contrast, a narrower distance distribution has been measured within one of the major helices, indicative of native-like secondary structure. The surface accessibility of all-Ala &alpha;-LA and that of two other variants ([28-111] &alpha;-LA and 4SS &alpha;-LA) has been probed using solvent PREs obtained using TEMPOL, a paramagnetic co-solute. This has revealed differences in the solvent-exposure of hydrophobic residues due to the removal of disulphide bonds. This method has also identified buried hydrophobic residues that contribute to forming the molten globule’s stable, native-like core.

La acetilcolinesterasa (AChE) hidroliza el neurotransmisor acetilcolina. La enzima se presenta en distintas formas moleculares. Tetrámeros hidrofílicos de AChE de suero fetal bovino se purificaron, sometieron a un tratamiento químico desnaturalizante o reductor, y estudiaron mediante análisis de sedimentación, cromatografía, fluorescencia intrínseca y unión de sondas hidrofóbicas. La transformación de los tetrámeros hidrofílicos en dímeros y monómeros anfifílicos demostró la alta flexibilidad conformacional de las subunidades de AChE, lo que podría ser crucial en la síntesis del conjunto completo de sus formas moleculares, y aportó una explicación de cómo algunas de sus formas interaccionan con las membranas. Para entender la heterogeneidad molecular de la AChE, también se empleó el anticuerpo AE-1, que interaccionó de forma desigual con oligómeros y monómeros de AChE de distintas fuentes. Experimentos de Western blot demostraron que el epítopo de AE-1 es de naturaleza confor macional. Finalmente, los datos experimentales descartaron la fosforilación de la AChE con proteína quinasa A. / Acetylcholinesterase (AChE) hydrolyzes the neurotransmitter acetylcholine. The enzyme exists in several molecular forms. AChE hydrophilic tetramers from fetal bovine serum were purified, chemically denatured or reduced, and studied by sedimentation analysis, hydrophobic chromatography, intrinsic fluorescence spectra and binding of amphiphilic probes. Conversion of the hydrophilic tetramers into amphiphilic dimers and monomers showed that AChE subunits possess a flexible conformation, which may be important for generating a full set of molecular forms, and gave an explanation of the interaction of certain AChE forms with membranes. Another approach to determine the molecular basis for the structural heterogeneity of AChE was to use the antibody AE-1, which distinctly reacted with AChE oligomers and monomers from different sources. The results of Western blot revealed that the determinant for AE-1 consisted of a conformational domain, not a primary sequence region. Finally, the experimental data rejected the phosphorylation of AChE at non-consensus protein kinase A sites.