Abstracts and Papers

Here you can find teacher abstracts and links to papers for the round table paper discussion sessions. Missing content will be added continuously, so come back later if you are lacking information.

Mikael Akke	Jan H Jensen	Lene Oddershede
Lise Arleth	Birthe B Kragelund	Kasper D Rand
Alessandro Borgia	Micha Ben Achim Kunze	Xavier Salvatella
John Christodoulou	Guillermo Montoya	Kaare Teilum

Mikael Akke

Biophysical Chemistry, Lund University

PROTEIN DYNAMICS IN MOLECULAR RECOGNITION

Conformational dynamics is intimately connected to molecular recognition involving proteins in two principal ways. Conformational dynamics contributes both to the kinetics and the thermodynamics of ligand binding. First, conformational transitions between different substates can control access to the binding site (kinetics). Second, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics).

In regard to binding kinetics, it is of great interest to understand the underlying protein dynamics and its role in setting the time scale for protein–ligand association into the final complex. A minimal framework for investigating ligand binding involves two different pathways: the induced-fit pathway and the conformational-selection (or select-fit) pathway, the relative importance of which has been the subject of recent investigations. I will describe results from NMR relaxation dispersion experiments that serve to map the conformational changes of a protein, as it moves across the energy landscape from the free state to the ligand-bound state.

Conformational entropy has been shown to contribute significantly to binding affinity, selectivity, and allostery. NMR relaxation experiments provide a unique probe of conformational entropy by characterizing fast bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side-chain order parameters, one can estimate the contributions from conformational entropy to the free energy of binding. This approach can be combined favorably with molecular dynamics simulations to provide increased coverage of the sampled degrees of freedom.

Papers

Lise Arleth

Niels Bohr Institute, University of Copenhagen, Denmark

X-RAYS AND NEUTRONS FOR STRUCTURAL AND DYNAMICAL STUDIES OF BIOLOGICAL MOLECULES

The lecture by Lise Arleth will provide a short overview of the general use of X-rays and neutrons in structural and dynamical investigations of biological molecules and a short overview of the present development within large scale facilities for X-rays and neutrons. A more elaborate overview will then be provided on the small-angle scattering (SAS) technique, which is one of the most popular techniques for obtaining structural information about biological molecules in solution. In particular, it will be discussed what can be obtained through, respectively, X-ray and neutron based small-angle scattering (SAXS and SANS).

The discussion in the afternoon workshop will focus on recent work and perspectives for the investigation of the structure, structural disorder and dynamics of water soluble proteins and membrane proteins through the reading of the three papers below.

Papers

Alessandro Borgia

Department of Biochemistry, University of Zurich, Switzerland

FLUORESCENCE SPECTROSCOPY AT THE SINGLE MOLECULE LEVEL: INVESTIGATION OF INTRINSICALLY DISORDERED PROTEINS (IDPS) AND MISFOLDING IN MULTIDOMAIN PROTEINS

Papers

John Christodoulou

Department of Structural and Molecular Biology, UCL, United Kingdom

PROTEIN FOLDING ON THE RIBOSOME

The folding processes of nascent chains are intricately linked to their chain elongation, which occurs in a vectorial manner as the N-terminal part of the nascent chain emerges from the ribosome. While increasingly detailed pictures of the structures of ribosomes are emerging, little is known at the atomic level about the structural and co-translational folding properties of nascent polypeptide chains. The use of NMR spectroscopy on ribosomes and ribosome nascent-chain complexes (RNCs) is providing detailed structural insights of the conformations of protein chains while they are being created on the ribosome. By producing in-vivo derived RNCs in which the nascent polypeptide is selectively isotopically-labelled, recent work has allowed us to use NMR to follow co-translational folding processes at a residue-specific level.

We have studied RNCs of a tandem of immunoglobulin-like domains where NMR reveals a folded N-terminal domain and a disordered but compact C-terminal domain. Most significantly, in order to study how Dom5 acquires its native structure co-translationally, we shortened the RNC constructs by reducing the length of FLN6. We found that the ribosome modulates folding, as the complete Dom5 sequences emerges well beyond the tunnel before acquiring native structure, while in isolation it folds spontaneously, even when truncated. This suggests that regulating structure acquisition during biosynthesis may reduce the probability of misfolding, particularly as homologous domains emerge from the ribosome. We compare these findings to those of a model nascent chain system of misfolding prone, alpha-synuclein.

Our recent developments in the use of NMR to produce the first experimentally derived structures of ribosome nascent chain folding will be discussed. These combine data on the same samples from NMR, including RDCs and cryo electron microscopy. These studies are allowing us to describe structures sampled during the vectorial emergence, the interactions of the emerging chains with the ribosome and also how the molecular chaperone, the trigger factor interacts with the nascent chain.

Papers

Jan H. Jensen

Department of Chemistry, University of Copenhagen, Denmark

PREDICTING PH-DEPENDENT PROPERTIES OF PROTEINS

In my talk I'll introduce the program Propka (propka.org) which can predict the pKa values of ionizable groups in proteins and protein-ligand complexes from the protein structure. I'll also talk about how these pKa values can be used to predict pH dependent properties of proteins, such as the charge of an amino acid, isoelectric point, enzymatic activity, stability, and protein-ligand and protein-protein binding free energies

Papers

Birthe B. Kragelund

Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Denmark

INTRINSIC DISORDER IN MEMBRANE PROTEINS - ROLES IN REGULATION AND SCAFFOLDING WITH LINKS TO PHOSPHORYLATION

Intrinsically disordered regions (IDRs) in membrane proteins play central roles in cellular signaling processes and like their structured protein counterparts, they engage in interaction networks of regulatory nature. Intracellular domains of many membrane proteins contain large IDRs of importance for function and with numerous predicted as well as confirmed phosphorylation sites. Due to their lack of globular structure insight into their structure-function relations and their functional dynamics have been crucially lacking. In this lecture I will address how NMR spectroscopy, biophysics and cell-biology are used to decipher regulatory roles of protein intrinsic disorder in cytokine receptors and in ion transporters with direct links to their phosphorylation and to how membrane cross-talk is involved. The interplay of intrinsic disorder and phosphorylation in these proteins highlights specific space and temporal effects in scaffolding including interplay with some of the major signaling pathways such as JAK2/STAT and MAPK-signaling.

Papers

Micha Ben Achim Kunze

Department of Biology, University of Copenhagen, Denmark

USING EPR AND SITE-DIRECTED-SPIN-LABELING AS A TOOL TO ELUCIDATE STRUCTURAL ENSEMBLES OF PROTEINS

The lecture will give a brief overview of how EPR and SDSL can provide structural information of proteins independent of the proteins size. Starting with short introduction of the basic method, the majority of the session will focus on examples of how to use EPR and SDSL in structural biology: investigating small peptide:protein interaction [1], long coiled-coil structures [2] and large complexes such as bacterial secretion systems [3].

Finally, EPR will be compared to other similar techniques such as FRET and brought into context with more complementary methods.

Papers

Guillermo Montoya

Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark

Papers

Lene B. Oddershede

Niels Bohr Institute, University of Copenhagen, Denmark

DYNAMICS OF DNA ASSOCIATING PROTEINS

Most biological functions of DNA rely on interaction between site-specific proteins and DNA. The proteins must first find their targets, then attach to DNA, and thereafter perform their job. In cells, DNA is constantly twisted, bent, stretched or even fragmented or re-connected by numerous proteins mediating genome transactions. A physical characterization of DNA is important to understand DNA-protein interaction, and the presentation will begin by exploring the mechanical properties of DNA, how DNA reacts to stretch and twist, by employing optical tweezers as a measurement tool. The interaction of DNA and proteins rely on the physical state of the DNA, and such interaction is often more clearly revealed in a single molecule assay as ensemble averaging often masks dynamics. To understand the dynamics and interaction between naturally supercoiled DNA and DNA associating proteins, a novel assay was invented where supercoiled circular DNA plasmids were individually tethered by peptide nucleic acid (PNA) handles. This tethered plasmid single molecule assay revealed that, compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch were significantly increased in the supercoiled system compared with a linear assay, thereby pinpointing the importance of supercoiling for DNA associating protein dynamics. Another example of the dynamics of DNA associating proteins presented during the talk, is the role of the XXRCC4-XLF complex for connecting DNA fragments during non-homologous end joining during repair of DNA double-strand breaks in mammalian cells.

In the discussion session focus will be on mechanical characterization of biopolymers using optical tweezers and all participants are kindly asked to bring their computers for the discussion session.

Papers

Kasper D. Rand

Department of Pharmacy, University of Copenhagen, Denmark

PROBING PROTEIN DYNAMICS BY HYDROGEN-DEUTERIUM EXCHANGE AND MASS SPECTROMETRY

The role of subtle changes in protein mobility and structural flexibility in the regulation of protein function is becoming increasingly evident.

Sensitive, high-resolution techniques are needed to detect the elusive dynamic interplay of distant sites critical for protein function. The hydrogen/deuterium exchange (HDX) of main-chain amides is highly sensitive to dynamic changes in conformation between protein states, and report on the overall flexibility of the protein backbone and local hydrogen bonding (i.e. conformational dynamics).

Mass spectrometry (MS) has evolved to be a powerful technique to measure protein HDX and thus monitor protein dynamics in solution, due to tolerance to complex protein systems, buffer composition and low sample concentration. More recently, the integration of electron transfer dissociation (ETD) into the HDX-MS workflow has enabled the mapping of conformational changes in proteins at a spatial resolution own to individual residues.

This lecture will give an overview of how to measure the hydrogen/deuterium exchange of proteins by mass spectrometry and how this information can be used to detect and map conformational differences between functional protein states or protein-ligand complexes.

Papers

Xavier Salvatella

Department of Chemistry and Molecular Pharmacology, IRB Barcelona, Spain

DISORDER TO ORDER TRANSITIONS IN THE REGULATION OF TRANSCRIPTION: BIOMEDICAL IMPLICATIONS

The initiation of gene expression relies on transient protein protein interactions between the transactivation domains of transcription factors and the basal transcription machinery. Such transactivation domains are in general intrinsically disordered and their affinity for the transcription machinery and, as a consequence, their ability to activate transcription can be modulated by changes in their propensity to adopt secondary structure as well as by post translational modifications. In my laboratory we are very interested in understanding how the transcription factor androgen receptor activates transcription because inhibiting this process represents a powerful therapeutic approach to halt the progression of prostate cancer that has become refractory to hormone therapy, the first line treatment for this disease. In my lecture I will present our current understanding of this topic, which we have obtained by using nuclear magnetic resonance as well as cell biology techniques.

Papers

Kaare Teilum

Department of Biology, University of Copenhagen, Denmark

CU2+ BINDING TO FIBRILLATED PROTEIN MITIGATES THE PRODUCTION OF REACTIVE OXYGEN SPECIES

Elevated levels of Cu2+ and other redox active metal ions have been found in the senile plaques from Alzheimers disease patients and in the Lewy bodies and cerebrospinal fluid of Parkinsons disease patients. It is not well understood how the protein bound metal ions affect the progression of the diseases, but elevated levels of reactive oxygen species (ROS) are associated with both diseases. In this lecture, I review the current knowledge of how metal ions bound to fibrillated protein may be involved in ROS production and show examples of how Cu2+-protein complexes lowers the redox activity of the metal-ion and apparently mitigates the direct ROS production.