Teachers Abstracts – Section for Biomolecular Sciences - University of Copenhagen

Summer School 2009 > Teachers Abstracts

Talk titles and materials for round table discussions:

To be updated...... 

Bruce Palfay 

Title of talk and Round table discussion: Flavoproteins that play with fire but don't get burned. Safety through confirmational control.

Flavin prosthetic groups react in many ways. However, a particular flavoenzyme limits the reaction paths to those that accomplish the physiological objective of the enzyme; wasteful and toxic side-reactions are generally prevented by the protein. Many reduced flavoenzymes react with O2 to make flavin hydroperoxide intermediates. The enzymebound hydroperoxide is then used in reactions similar to those in synthetic organic chemistry. In some enzymes, the hydroperoxide reacts as an electrophile to hydroxylate nucleophiles, while other enzymes use the intermediate as a nucleophile to hydroxylate electrophilic substrates. Flavin hydroperoxides generated in aqueous solution in the absence of enzymes have lifetimes of microseconds – too short to allow meaningful biochemistry – because of the facile solvent-catalyzed elimination of H2O2. Flavoprotein hydroxylases prevent this, but still must allow the substrate access to the hydroperoxide. Two classes of flavoprotein hydroxylases illustrate very different strategies for ensuring that the hydroperoxide is used productively to hydroxylate substrates without wasting the reduced pyridine nucleotides used to generate the hydroperoxide and generating toxic H2O2. The aromatic hydroxylases do not allow the reaction of NAD(P)H unless a substrate is bound and ready for reaction. In contrast, the FMO family of hydroxylases and Bayer-Villiger hydroxylases protect the intermediate until substrate is encountered. These two contrasting strategies rely on controlling conformational changes. Details have become available for a few well-studied model systems from complementary transient kinetic, crystallographic, and mutagenesis studies. The interplay between these experimental approaches in elucidating these control strategies are illustrated in the three selected papers.

Alfieri, A., Malito, E., Orru, R., Fraaije, M.W., Mattevi, A. (2008) Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase. PNAS

Palfey, B.A., Moran, G.R., Entsch, B., Ballou, D.P., Massey, V. (1999) Substrate Recognition by "Password" in p-Hydroxybenzoate Hydroxylase. Biochem.

Frederick, K.K., Palfey, B.A. (2005) Kinetics of protein-linked Flavin Conformational changes in p-Hydroxybenzoate Hydroxylase. Biochem.

Bente Vestergaard

Title of talk: Structural analysis of protein suprastructure using solution SAXS - an
introduction to the method and selected examples.

Round table discussion: Applying SAXS to structural analysis in solution - complementarity to other structural methods, challenges and limitations.

Maxim V. Petoukhov and Dmitri I. Svergun. Analysis of X-ray and neutron scattering from biomacromolecular solutions

K. Nørgaard Toft, Bente Vestergaard, Søren S. Nielsen, Detlef Snakenborg, Mads G. Jeppesen, Jes K. Jacobsen, Lise Arleth, and Jörg P. Kutter (2008) High-Throughput Small Angle X-ray Scattering from Proteins in Solution Using a Microfluidic Front-End. Anal. Chem

Bente Vestergaard, Minna Groenning, Manfred Roessle, Jette S. Kastrup, Marco van de Weert, James M. Flink, Sven Frokjaer, Michael Gajhede, Dmitri I. Svergun (2007) A Helical Structural Nucleus Is the Primary Elongating Unit of Insulin Amyloid Fibrils. Plos Biology

Clive Bagshaw

Talk title: Identifying protein states using kinetic methods.

Round Table Discussion: Correlating structural staes and kinetic states.

Málnási-Csizmadia, A., Pearson, D.S., Kovács, M., Woolley, R.J., Geeves, M.A., Bagshaw, C.R. (2001) Kinetic Resolution of a conformational transition and the ATP Hydrolysis step using relaxation methods with Dictyostelium Myosin II mutant containing a single tryptophan residue. Biochem.

James, L.C., Tawfik, D.S. (2005) Structure and kinetics of a transient antibody binding intermediate reveal a kinetic discrimination mechanism in antigen recognition. PNAS

English, B.P., Min, W., van Oijen, A.M., Lee, K.T., Luo, G., Sun, H., Cherayil, B.J., Kou, S.C., Xie, X.S. (2006) Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nat Chem Bio

Dorothee Kern

Title of talk and Round table discussion:  The choreography of an enzyme’s dance - molecular pathways of conformational transitions in proteins

The synergy between structure and dynamics is essential to the function of biological macromolecules. While this is a widely accepted concept, we want to address one of the key challenges for any protein: How can a protein be dynamic and interconvert among folded substates for biological function but avoid unfolding at the same time?
Recent advances in technology development including NMR relaxation experiments allow us to characterize the energy basins in an energy landscape, the valleys in the mountains. The concept of pre-existing low populations of conformational substates which are essential for protein function, and the concept of a population shift via selected ligand binding, and not induced fit, is now becoming widely accepted. However, this concept raises the key subsequent question of how proteins can convert from one folded structure to another, how they climb over the mountain (the energy barrier) from one valley to another. This new question of the molecular pathways for conformational transitions will be addressed in this talk.
We quantitatively characterize the energy landscape of a signaling protein using NMR and molecular dynamics simulations. Importantly, we will correlate dynamics within the folded space with unfolding.  These results shed light into the question of how folded proteins can interconvert very efficiently among folded substates that require extensive rearrangements but avoid unfolding. Second, the energy landscape of an enzyme both during catalysis and in the absence of substrates is being characterized, which allows identification of dynamics that are linked to enzyme catalysis. Using a combination of high-pressure NMR experiments and computation, the transition pathway and in particular information about the transition-state can be inferred. Both examples illustrate the power of combining computation with quantitative NMR experiments and stopped flow fluorescence to unravel not only the states that are sampled by the protein but also the pathways of conformational transitions.
 In the round table discussion I will try something new and (hopefully) exciting (since we will talk about the excited state): You will play the referee in the controversy of contradictory transition pathways proposed for the same signaling protein by different groups. 

Latzer, J., Shen, T., and Wolynes, P. G., Conformational switching upon phosphorylation: a predictive framework based on energy landscape principles. Biochemistry 47 (7), 2110 (2008).

A. K. Gardino, J. Velos, M. Lei, A. Kivenson, C. Feng Liu, E. Z. Eisenmesser, W. Labeikovsky, M. Wolf-Watz, and D. Kern, Native-State Energy Landscape Reveals Activation Pathway in a Signaling Protein (2009)

Katherine Henzler-Wildman & Dorothee Kern (2007) Dynamic personalities of proteins. Nature Reviews

Gideon Schreiber

Title of talk and Round table discussion: Protein-Protein association

Schreiber, G., Haran, G., Zhou, H.-X. (2009) Fundamental aspects of Protein-Protein association kinetics. Chem. Rev.


Jane Clarke

Talk title: Folding and Mechanical Stability of Multidomain Proteins.

Round table discussion: The evolution of multidomain proteins and problems associated with misfolding in multidomain proteins.

Most protein folding studies involve small, single-domain proteins or, so-called “independently folding” domains cut from larger proteins.  However, most proteins (between 65 and 80% of all eukaryotic proteomes) contain more than one domain. The multidomain proteins we study in my laboratory experience significant stress in vivo. In my lecture I will describe some of our studies of the folding of multidomain proteins emphasising the experimental techniques we have used to dissect the folding of domains in a multidomain context. This will involve discussion of determination of protein stability (thermodynamics), protein folding kinetics and pathways and single molecule atomic force microscopy (AFM). In the Round Table Discussion we will also address the evolution of multidomain proteins and problems associated with misfolding in multidomain proteins. 

References:  The first two papers are reviews that summarise some of the topics, which will be discussed in the lecture and the Round Table session.  The last is an AFM paper, which shows that proteins may behave differently under mechanical stress.

 

Han, J.H., Batey, S., Nickson, A.A., Teichmann, S.A. & Clarke, J. (2007) The folding and evolution of multidomain proteins. Nature Reviews Mol. Cell Biol. 8, 319-330


Batey, S., Nickson, A.A. & Clarke, J. (2008) Studying the folding of multidomain proteins. HFSP J., 2, 295-415

Randles, L.G., Rounsevell, R.W.S. & Clarke, J. (2007) Spectrin domains loose cooperativity in forced unfolding. Biophys. j. 92, 571-577

 

J. Preben Morth

Title of talk and Round table discussion:  Crystal Structur of the sodium-potassium pump and the complete functional cycle for the P-type ATPases

The Na+,K+-ATPase, the sodium-potassium pump, was first described in 1957 by Jens C. Skou [1] - a discovery for which he was awarded the Nobel prize in Chemistry in 1997. The Na+,K+-ATPase belongs to the P-type ATPase family, and via formation and break-down of phosphoenzyme intermediates it derives the energy from ATP hydrolysis to pump Na+ out of the cell and K+ into the cell, thereby energizing the plasma membrane with steep electrochemical gradients for these key cations.The Na+,K+-ATPase is a hetero-trimeric complex composed of an a, b and g chain that all contain transmembrane segments. Na+,K+-ATPase from outer medulla in pig kidney was purified and solubilized in the presence of native lipids by the detergent C12E8 in a buffer containing MgF42- and Rb+ (a K+ congener) thus locking the enzyme in an occluded state mimicking a post-state of dephosphorylation. This preparation is stable for weeks. Three-dimensional crystals were grown by vapour diffusion. The crystals appear after 3-6 days and grow as thin plates to maximum dimensions of 25x100x400 mm or longer over a few weeks. SDS-PAGE analysis of redissolved crystals shows that they contain both the a, b chains, and two isoforms (A,B) of the g chain in kidney. No deglycosylation was required to obtain the crystals. A complete native dataset was obtained at 3.5 Å resolution on the X06SA beam line at the Swiss Light Source (SLS). The brilliant light source present at SLS was necessary to obtain useful data from these very weakly diffracting crystals. A low resolution molecular replacement solution allowed us to identify two heavy-atom derivatives (Ta6Br122+ and orange-Pt) by difference-Fourier analysis forming the basis for MIRAS phasing at 7 Å resolution. The crystal form has 75% solvent and contains two-fold NCS. Careful density modification with NCS and inter-crystal averaging was applied and extended the MIRAS phases to 3.5 Å resolution thus allowing for model building and refinement of the structure.

This the first structure of the Na+,K+-ATPase in the K+/Rb-bound form contains a nearly complete model of the a-subunit and shows the location of the transmembrane helices of the b and g subunits associated with a. Two strong peaks in the anomalous difference Fourier map pinpoint the position of  two occluded Rb+ sites (for K+), located between the transmembrane helices M4, M5 and M6. Structural features hint at a regulatory function interconnected with three putative Na+ sites for the Na+-bound forms. The extracellular part of the b subunit could not be traced, but our maps indicate that it represents a large, curved b-sheet like structure which covers most of the extracellular loops of the a-chain. The complete transport model for the Ca2+ -ATPase will be discussed, with basis in the struturel determination of the last functional step in the transport cycle, the E2P state, with BeF3-  mimicking the covalently bound phosphate group on the Asp351 [3]. 


C. Olesen, M. Picard, A.M. Winther, C. Gyrup. J.P. Morth, C. Oxvig,  J.V. Møller, P. Nissen, The structural basis of calcium transport by the calcium pump, Nature 450 (2007) 1036-1042.

Supplementary material to Olesen et al.

Supplementary video to Olesen et al. (opens in Quicktime)

 

J.P. Morth, B.P. Pedersen, M.S. Toustrup-Jensen, T.L. Sørensen, J. Petersen, J.P. Andersen, B. Vilsen, P. Nissen, Crystal structure of the sodium-potassium pump, Nature 450 (2007) 957-959.

Supplementary material to Morth et al.

Suplementary video to Morth et al. (opens in Firefox or other webbrowser with Flashplayer)

 


Kaare Teilum and Birthe B. Kragelund

Title of talk and Round table discussion: Entropy and flexibility in protein interactions

Teilum, K., Olsen, J.G., Kragelund, B.B. (2009) Functional aspects of protein flexibility. Cell Moll Life Sci

Frederick, K.K., Marlow, M.S., Valentine, K.G., Wand, A.J. (2007) Conformational entropy in molecular recognition by proteins. Nature

Jomain, J.-B., Tallet, E., Broutin, I., Hoos, S., von Agthoven, J., Ducruix, A., Kelly, P.A., Kragelund, B.B., England, P., Goffin, V. (2007) Structural and thernodynamic bases for the design of pure prolactin receptor antagonists. J Bio Chem.

Martin Blackledge

Title of talk: Characterization of Conformational Dynamics in Folded and Partially Folded Proteins using NMR Residual Dipolar Couplings

Round Table Discussion: Structural Behaviour of Unfolded and Partially Folded Proteins using NMR Residual Dipolar Couplings

Highly Populated Turn Conformations in Natively Unfolded Tau Protein Identified from Residual Dipolar Couplings and Molecular Simulation. M. D. Mukrasch, P.R.L. Markwick, J. Biernat, M. von Bergen, P. Bernado, C. Griesinger, E. Mandelkow, M. Zweckstetter and M. Blackledge  J.Am.Chem.Soc. 129, 5235-5243 (2007).

Mapping the Conformational Landscape of Urea-Denatured Ubiquitin using Residual Dipolar Couplings. Sebastian Meier, Stephan Grzesiek and Martin Blackledge J.Am.Chem.Soc. 129, 9799-9807 (2007).

Quantitative Conformational Analysis of Partially Folded Proteins from Residual Dipolar Couplings : Application to the Molecular Recognition Element of Sendai Virus Nucleoprotein. Malene Ringkjøbing Jensen, Klaartje Houben, Ewen Lescop, Laurence Blanchard, Rob W.H. Ruigrok and Martin Blackledge. J.Am.Chem.Soc. 130, 8055-8061 (2008).

Rolf Hilgenfeld

Title of talk and Round Table discussion: Protein crystallography and protein dynamics/instrinsic disorder

Coronaviruses have the largest RNA genome known (up to 31 kb of single-stranded, positive-sense RNA). The replication/transcription machinery of these viruses is encoded by open reading frame 1, which comprises no less than 2/3 of the entire genome. It is transcribed and translated into two huge polyproteins, pp1a (450 kD) and pp1ab (790 kD). Translation of the latter requires a (-1) ribosomal frameshift. The polyproteins are processed by one or two papain-like proteases (PLpro) and a chymotrypsin-like main protease (Mpro, Nsp5) that carries a Cys...His catalytic dyad in its active site. The 16 non-structural proteins (Nsp’s) thus created assemble at double-membrane vesicles derived from the endoplasmic reticulum, to form the viral replicase/transcriptase complex (RTC). Through membrane-spanning segments in Nsp3, Nsp4, and Nsp6, the RTC is anchored to the membrane. By X-ray crystallography and NMR spectroscopy, the three-dimensional structures of several Nsp’s embedded in pp1a have been determined. In some cases, hypotheses suggesting specific functions could be developed on the basis of the structures. Remarkably, many of the structures revealed a new fold and the presence of disulfide bonds, which are rare in cytosolic proteins. Also, conformational changes have been observed depending on the pH and redox characteristics of the environment, and several of the proteins contain regions predicted to be intrinsically unfolded. The presentation will introduce the concept of intrinsically unfolded proteins.

J.H. Fong et al. (2009).: Intrinsic disorder in protein interactions: Insights from a comprehensive structural analysis. PLoS Comput. Biol.

Y. Zhai et al. (2005): Insights into SARS-CoV transcription and replication  from the structure of the nsp7-nsp8 hexadecamer. Nature Struct. Mol. Biol. 12, 980 - 986

Thomas Kiefhaber

Title of talk and Round table discussion: Characterization of protein folding barriers by rate-equilibrium free energy relationships

Cho, J-H., Raleigh, D.P. (2006) Denatured state effects amf the origin of nonclassical ø Values in protein Folding. J Am Chem Soc

Sanchez, I.E., Kiefhaber, T. (2003) Origin of unusual ø-values in protein folding: Evidence against specific nucleation sites. J Mol Bio

Sanchez, I.E., Kiefhaber, T. (2003) Hammond behavior versus ground state effects in protein folding: Evidence for narrow free energy barriers and residual structure in unfolded staes. J Mol Bio

Vern L. Schramm

Title of talk and Round table discussion: Enzymatic Transition States, Analogues and Dynamics.

Vern L Schramm (2005) Enzymatic transition states and transition state analogues. Current Opinion in Structural Biology

Andrew S. Murkin, Peter C. Tyler, and Vern L. Schramm (2008) Transition-State Interactions Revealed in Purine Nucleoside Phosphorylase by Binding Isotope Effects. J. Am. Chem. Soc.

Suwipa Saen-oona, Sara Quaytman-Machledera, Vern L. Schrammb, and Steven D. Schwartza (2008) Atomic detail of chemical transformation at the transition state of an enzymatic reaction. PNAS