FUNCTIONAL AND STRUCTURAL INSIGHTS INTO
The FEZ1 protein homo-dimerizes via a disulfide Bond
Furlan AS1, Alborghetti, MR2, Silva, J.C.3, Paes Leme, A.F.1, Sfor¸ca, ML1, Torriani, I.1, Zeri, A.C.; Zeri, A; Zeri, A.C.M.1, and Kobarg, J.1
1 Laborat´orio Nacional de Luz S´ıncrotron - Campinas SP Brazil
2 Centro de Biologia Molecular Estrutural - Campinas SP Brazil
3 Universidade Estadual de Campinas - Campinas SP Brazil
The human proteins FEZ1 (fasciculation and elongation protein zeta 1) and FEZ2 are orthologs of the protein UNC-76 from C. elegans, involved in the growth and fasciculation of the axon. Pull down assays showed that the protein FEZ1 interacts with other proteins (e.g. SCOCO), mitochondria and vesicles, via its C-terminal region. These components may therefore represent cargoes to be transported along the microtubule, and the transport may be mediated by FEZ1 association to ki- nesins (KIF3A). We previously showed that FEZ1 dimerizes in its N-terminal region and interacts through its C-terminus with other proteins. Here we study the frag- ment FEZ1(92-194) to verify the prediction for a coiled-coil region in this region and the formation of a probable disulfide bond through Cys-133, both of which are possibly important to mediate the proteins homo-dimerization. We obtained puri- fied samples of FEZ1(92-194) and performed native acryl amide gel electrophoresis under reducing or non-reducing conditions. Furthermore, we performed Small An- gle X-ray Scattering (SAXS) measurements, where we confirmed that the molecular mass of the non-reduced sample (dimer) was exactly twice the mass of the TCEP- reduced one. (monomer). We are starting to perform Nuclear Magnetic Resonance analyses, which so far suggest the a strucutrured region of about 15 amino acids in the N-terminal region of FEZ1(92-194). Finally, a comparative mass spectrometry analysis of reduced and non-reduced protein samples showed that the cysteine could be oxidized in the full-lenght FEZ1 protein. In summary, our results suggest that FEZ1 dimerizes via its N-terminal through a disulfide bond and coiled-coil inter- actions. The disulfide bond may be important for FEZ1 role as a bivalent adaptor molecule, since it establishes a strong link between the monomers. Our model sug- gests that the FEZ1 C-terminal can be divided in two regions: one may interact with microtubule associated proteins (tubulin, KIF3A, CLASP2 are FEZ1 interac- tors) while the other one may interact with candidate cargoe proteins (SCOCO), vesicles and mitochondria.
SAXS studies of DNA self-assembled structures
Oliveira, C. L. P1, Pedersen, J.S.1, Andersen, F. F.1, Knudsen, B. R.1, Irish, E.2, Labean, T.2, Andersen, E. S.1, and Kjems, J.1
1 Aarhus University - Aarhus Denmark
2 Duke University Medical Center - Durham CN United States of America
For over 20 years, DNA has been recognized as a useful construction material for nanotechnology because of its readily programmable molecular recognition and pre- dictable local geometry. Many artificial, self-assembled DNA nanostructures have been reported using various geometric structures and functionalities, including one- and two-dimensional periodically patterned structures, three-dimensional polyhe- dra, DNA computers, and mechanical devices. The visualization and analysis of these structures is a very important issue, in order to confirm and judge the con- formation of the formed structures. Electron microscopy techniques are one of the major tools in this respect permitting the direct visualization of the structures in real space. However, one of the intrinsic limitations of this technique is the require- ment for fixation and staining of the samples, which both might lead to artefacts.
On other hand, the small angle X-ray scattering (SAXS) technique permits the study of the structure in solution giving information on a large ensemble of the structures simultaneously. We performed SAXS experiments on self-assembly 2D (’tiles’ and ’tracks’) and 3D DNA structures (cages and boxes). For the data anal- ysis new modelling methods had to be developed in order to take into account the expected overall geometry of the structures, the DNA strand base pairing, and the known structural elements. Employing several modelling approaches [1,2] we have obtained the three-dimensional structures, structural dimensions and polydisper- sity levels for the studied DNA self-assembled systems in solution.
References
[1] F. F. Andersen et al. 2008 Nucleic Acids Research., 36, 1113.
[2] E. S. Andersen et al. 2009 Nature 459(7243), 73-76.
Acknowledgements:
A in situ SAXS Study of Glucagon Fibrillation
Oliveira, C. L. P1, Behrens, M. A.1, Pedersen, J. S.1, Erlacher, K2, Otzen, D. E.1, and Pedersen, J.S.1
1 Aarhus University - Aarhus Denmark
2 Bruker BioSpin GmBH - Rheinstetten Germany
Protein amyloid formation proceeds through a number of different stages. Oligomeric species observed at early stages have aroused particular interest because of evidence for their involvement in cytotoxic processes such as membrane permeabilization.
It is unclear whether these oligomers are obligate precursors to fibrils or represent dead-end species which impede fibrillation. Because of the many interconverting species present during amyloid formation, it is important to study the process as non-invasively as possible. Small angle X-ray scattering (SAXS) measurements allow us to monitor structural changes in solutionin situfor a population of differ- ent species over time. Here [1] SAXS was used to provide a detailed time-resolved structural description of the in situ fibrillation of the 29-residue peptide hormone glucagon at pH 2.5 from monomer and early oligomers to mature fibers. Investi- gation of the pseudo-equilibrium behavior in the lag phase prior to fibrillation at several concentrations showed that glucagon can be found as monomers, dimers, trimers or hexamers in the concentration range from 1 10 mg/ml. After the lag phase, a short rod-like protofibril is formed and subsequently grows in length and assembles into long triple bundled mature fibers. Applying several newly developed modeling tools to the experimental data the full process, from early oligomerization stages up to the mature fiber formation could be described as associations between glucagon molecules. The modeled fibrillation process is described in Figure 1. We propose that on-pathway fibrillar intermediates, which accumulate substantially during fibrillation at high protein concentrations, share this elongated shape which easily allow them to be incorporated into mature fibrils. This contrasts with the annular shape that is suggested to be involved in cytotoxic membrane permeabi- lization and which may represent a dead-end species off the fibrillar pathway.
Reference
[1] C. L. P. Oliveira et al. 2009 J. Mol. Biol., 387, 147.
Acknowledgements:
Structural studies of Intrinsically Unstructured Proteins by Small-Angle X-ray Scattering
Silva, J.C.1, Lanza, D.C.F1, Bressan, G.C.2, Kobarg, J.2, and Torriani, I.L.1
1 Universidade Estadual de Campinas - Campinas SP Brazil
2 Laborat´orio Nacional de Luz S´ıncrotron - Campinas SP Brazil
The tenet that a three-dimensional protein structure dictates its biological role has been defied by the increasing evidence of natively unstructured proteins which display functions that require conformational disorder. For example, membrane transport, molecular recognition, post-translational modifications, cellular signal- ing and regulatory mechanisms are important functions often accomplished by intrinsically unfolded proteins. For these reasons, there is a clear need for the de- velopment of approaches that lead to qualitative and quantitative characterization of flexible and intrinsically unstructured proteins in solution. Dealing with protein structure determination of intrinsically unstructured proteins (IUP), or natively unfolded proteins is a new challenge to be faced. A significant effort has already been directed toward developing empirical sequence based bioinformatic tools for disorder prediction in the IUP structures. Nonetheless, much of the progress on protein structure characterization has been achieved mainly with macromolecules with well-defined and rather rigid tertiary structures. Structural analyses of flex- ible macromolecules almost exclude high-resolution techniques. In such cases, the Small-Angle X-ray Scattering (SAXS) technique can efficiently reveal the spatial conformation, the organization of protein domains, including disordered and un- known parts of the molecule, and allows the study of conformational changes.
SAXS provides important dimensional parameters of the protein conformation and a quantitative analysis on the degree of compactness (degree of folding) of an IUP.
A new approach, employing ensemble optimization, has been specially developed to analyze flexible structures. In this work we have explored all sorts of strategies to single out the range of possibilities of IUP structural characterization provided by the SAXS technique. Some of our studies on two human IUPs are presented.
We have studied the Fasciculation and Elongation Zeta-1 protein (FEZ1) which is related to axon growth and may be related to schizophrenia, and the human protein calledKi-1/57 whose functional available data suggests its complex role in
SAXS studies of flexible multidomain proteins
Silva, J.C.1, Borges, J.C.2, Quaresma, A. J. C.3, Bressan, G.C.3, Kobarg, J.3, Ramos, C.H.I.1, and Torriani, I.L.1
1 Universidade Estadual de Campinas - Campinas SP Brazil
2 Instituto de Quimica de Sao Carlos - S˜ao Carlos SP Brazil
3 Laborat´orio Nacional de Luz S´ıncrotron - Campinas SP Brazil
Proteins with well structured domains linked by flexible regions are generally in- volved in important biological functions. These structural features are often ob- served in proteins that interact with DNA or RNA and in some chaperones. From the point of view of high resolution structure solving, these proteins pose a real challenge when NMR or crystallographic methods are employed. The existence of flexible regions in the molecules is sometimes a real obstacle. TheSmall Angle X- ray Scattering (SAXS) provides low resolution data that in many instances allows the investigation of important details in biological structure-function key problems.
SAXS can efficiently reveal the spatial conformation, the organization of protein domains, including disordered and unknown parts of the molecule, and allows the study of conformational changes. Moreover, the most potentially useful outcome of this technique is the possibility of performing a low-resolution modeling of the protein. These modeling methods allow us to restore the conformational space as well as the possible conformations adopted by flexible multidomain proteins in so- lution. Ab initio methods, using the well knowndummy atoms (DA) anddummy residues (DR) routines, lead to the calculation of the low-resolution models of proteins in solution. The DR-based method has the advantage of being chain-like compatible and considers the number of residues known a priori from the amino acids sequence. For flexible multidomain proteins, there is still therigid body mod- eling method, which combines high resolution of the domains and simulated flexible loops of unknown structures linking them. In this work we describe the results of the structural analysis of two types of multidomain proteins: the deleted-domain mutants of the protein HSP40 Sis1, whose functional activity is under study, and one heterogeneous nuclear protein from family Q1 (hnRNP Q), which is related to a wide array of RNA important functions.
Acknowledgements: FAPESP, CNPq, LNLS, CeBiME