«Book of Abstracts ssNMR in Comparison to Other Structural Investigation Techniques Beat Meier Department of Chemistry and Applied Biosciences, ETH ...»
NMR Meets Biology
Book of Abstracts
ssNMR in Comparison to Other Structural Investigation
Department of Chemistry and Applied Biosciences, ETH Z¨ rich, Switzerland
The lecture will show how solid-state NMR can be combined with other structural methods,
notably electron microscopy and x-ray crystallography. After an introduction to the topic, we
will show this at the example of the ASC-PYD ﬁlaments, as well as the DnaB Helicase and its interaction partners.
Date: 14 January 2016 Time: 16:00-17:00 Future Challenges in Structural Biology Hartmut Oschkinat Leibniz-Institut f¨ r Moleculare Pharmakologie, Berlin, Germany u Abstract Date: 14 January 2016 Time: 17:30-18:30 Practical Aspects of Solid State NMR Gerhard Althoﬀ Bruker BioSpin GmbH, Silberstreifen, Germany Abstract This contribution focusses on the hardware needed for of solid-state NMR. As the vast majority of solid-state NMR experiments is performed under magic angle sample spinning (MAS) the
emphasis is on MAS probes with respect to:
• MAS rotor and stator design
• Properties of air bearings
• Ancillary eﬀects of sample spinning: frictional heating and centrifugal forces
• Rotor handling and precautions
• Probe RF design for double and triple resonance experiments
• Probe channel insulation and external ﬁlters
• Ancillary eﬀects of RF pulses: RF sample heating and coil designs to minimize this eﬀect
• Special probe designs for low or high temperatures and for dynamic nuclear polarization (DNP) Date: 14 January 2016 Time: 18:30-19:30 The Hamiltonian Operator Beat Meier Department of Chemistry and Applied Biosciences, ETH Z¨ rich, Switzerland u Abstract The basics of the quantum description of NMR spectra using spherical tensor operators will be introduced and applied to static and MAS experiments in two and three dimensions.
Date: 15 January 2016 Time: 08:00-09:20 Exercise Hour
Abstract Generating diﬀerent eﬀective Hamiltonians during various time periods of experiments is one of the key reasons for the large number of experiments that can be realized in solid-state magicangle spinning NMR. Although the laboratory-frame spin-system Hamiltonian is time independent, magic-angle spinning as well as radio-frequency irradiation lead to a time dependence of the Hamiltonian. Therefore, methods to treat time-dependent Hamiltonians are essential for understanding solid-state MAS NMR experiments. In this lecture the origin of time-dependent Hamiltonians as well as the two main ways to treat them, namely average Hamiltonian theory and Floquet theory, are discussed. Simple examples will be used to illustrate and compare the two methods.
Date: 15 January 2016 Time: 11:30-13:00
Abstract In most forms of NMR we deal with nuclear spin systems which are very close to thermal equilibrium. However there are several phenomena which involve highly non-equilibrium nuclear spin systems, and which are of great interest since they may be associated with strongly enhanced NMR signals, sometimes by many orders of magnitude. These are: (1) hyperpolarization eﬀects, in which samples are prepared with nuclear spin temperatures which may be much smaller than that of the environment; (2) spin isomerism, which entangles the spatial and spin quantum states, investing some nuclear spin states with energies which greatly diﬀer from ordinary nuclear Zeeman energies, and which allows the ready preparation of samples in highly non-equilibrium spin states. I expect to improvise a discussion of the various phenomena involving nuclear spins far from equilibrium, the relationship between them, and the ﬁeld of long-lived nuclear spin states.
AbstractMembrane proteins are highly dynamic molecules that undergo numerous dynamic conformational transitions with varying amplitudes within a broad window of correlation times. In the presentation, I will provide a summary on the dynamic modes of membrane proteins and an overview of solid-state NMR methods that allow to study these motions. From the general classiﬁcation of motions observed on the NMR time scale (fast (τ µs), intermediate (τ ∼ µs), slow motions (τ µs)) some well-established methods for the elucidation of membrane protein dynamics will be discussed. These include the study of motionally averaged anisotropic interactions detected in separated local ﬁeld experiments, relaxation time measurements and analysis, line shape analysis for intermediate time scale motional analysis, and exchange spectroscopy to study slow (millisecond timescale) protein.
International Centre for Genetic Engineering and Biotechnology, New Delhi, India
AbstractRNA binding proteins or RBPs play an indispensable role in cellular machinery, especially processes such as transcription, post-transcriptional modiﬁcation of RNA, RNA transport and stabilization, among others. Post-transcriptional modiﬁcations of RNA are a major route through which eukaryotes regulate gene expression. These modiﬁcations include splicing, mRNA polyadenylation, 5 capping and RNA editing. RBPs bind RNA through speciﬁc RNA binding domains (RBDs) or modules. Binding aﬃnities and speciﬁcities vary throughout this family of proteins.
The RNA recognition motif or RRM is the most widely distributed RBD in nature. RRMs are canonically identiﬁed through the presence of two RNA binding consensus motifs (RNP). They have a common three-dimensional architecture, which classically consists of a four-stranded βsheet supported by two α-helices, with the β-sheet serving as the major surface for RNA recognition.
In spite of these unifying traits of RRMs, they possess a remarkably diverse RNA recognition capability. Our recent delineation of the RNA-binding mechanism of human TAF15 protein highlights how the concave face of its carboxy terminal RRM recognizes structured loop elements on RNA, in a non-canonical manner. Mutations in this protein have been implicated in familial amyotrophic lateral sclerosis or FALS. The human ETR3 protein on the other hand, possesses three RRMs, which sequence-speciﬁcally recognizes CUG and UG-rich RNA. This is carried out through speciﬁc interaction of a uracil base by a cleft on the β-sheet surface. The N-terminal RRM of malarial SR1 alternative splicing factor that semi-speciﬁcally recognizes pyrimidines exhibits a similar mode of interaction. We have delineated how the latter two instances represent a canonical form of RRM-RNA interaction, with π − π interactions between aromatic amino acids and nucleotide bases being responsible for binding. On the other hand, the TAF15-RRMRNA interface is largely dominated by hydrogen bonding between charged amino acids and polar groups on the RNA. Our work on RNA recognition by RRMs thus paints an interesting picture of how a single fold is able to recognize diﬀerent cognate RNAs by virtue of minor but crucial alterations to its binding surface. In addition, delineation of RNA binding speciﬁcities of RRMs has provided molecular clues to the progression of debilitating diseases such as myotonic dystrophy, FALS and malaria.
AbstractUnderstanding the mechanisms of biological processes requires precise knowledge of the threedimensional structures of the executor molecules such as proteins, bioactive peptides and others.
Atomic-resolution structures of well-folded proteins or complexes can be obtained from X-ray crystallography. However, a large number of proteins or domains of large proteins (e.g., in signaling cascades) and bioactive peptides (e.g., antimicrobial peptides, amyloid peptide) appear to be dynamic, thus limiting the application of X-ray-based methods. On the other hand, gaining insights into such molecular systems at the atomic level is possible using nuclear magnetic resonance (NMR) spectroscopy. The functional aspect of neurodegenerative diseases linked with amyloid beta (Aβ40 / Aβ42 ) ﬁbril formation is well known from the in vitro studies. However, membrane could play an important role for this ﬁbril formation. To date, there is a paucity of information detailing the interaction of amyloid beta (Aβ40 / Aβ42 ) proteins with membrane structures. In this talk I shall focus on elucidating the biophysical properties of the interactions between amyloid beta (Aβ40 / Aβ42 ) proteins and model membrane system.
AbstractMAS NMR spectroscopy is an essential method for structural and dynamics analysis of biological systems and is particularly powerful for atomic-resolution studies of large biomolecular assemblies where other structural techniques provide limited information. In MAS NMR studies of biomolecular assemblies, sensitivity and resolution remain a main challenge. I will discuss the principles and applications of nonuniform sampling (NUS) as one approach for gaining bona ﬁde time-domain sensitivity enhancements. I will present an overview of theory and experiments demonstrating that with the use of appropriately designed NUS sampling schedules, sensitivity gains of 1.7 - 2 fold are attained without the compromise in resolution. These sensitivity enhancements are compounded in each indirect dimension, resulting in considerable time savings and enabling collection of nD MAS NMR spectra in systems where traditional linear sampling data collection is prohibitively time consuming. I will discuss the maximum entropy interpolation method (MINT) introduced by us for processing of NUS data that permits quantiﬁcation of sensitivity. I will demonstrate that NUS is compatible with paramagnetic-relaxation-assisted condensed data collection (PACC) under fast MAS conditions, a combined NUS-PACC approach, yielding dramatic, 20-26 fold time enhancements in heteronuclear-detected 3D experiments. In the second part of the lecture, I will discuss approaches for site-speciﬁc determination of heteronuclear dipolar, heteronuclear CSA, and 1 H CSA interactions. RN-symmetry based sequences originally introduced by Malcolm Levitt and colleagues, are a powerful framework in the design of experiments for recoupling desired anisotropic tensorial interactions. These experiments are ideally suited for site-speciﬁc measurements of dipolar and CSA tensors in biological systems, including biological assemblies, and are compatible with a broad range of MAS conditions, including spinning frequencies of 40 kHz and above. I will discuss the application of this approach to the determination of molecular dynamics in HIV-1 capsid assemblies through the measurements of backbone dipolar interactions, where, in conjunction with MD simulations, we have obtained a comprehensive view of the molecular motions. I will also discuss the measurements of 1 H CSA tensors in a microtubule-associated CAP-Gly protein, and the connection of the determined 1 H CSA parameters to the hydrogen bonding interactions.
Date: 16 January 2016 Time: 09:40-11:10
AbstractDeuterated proteins with diﬀerent degree of protonation have been used successfully used in tertiary structure determination of proteins by solid-state NMR. However, not all proteins can be deuterated. So, new methods are required to structurally characterize non-deuterated biomolecules. The progress in magic-angle spinning (MAS) technology now provides a possibility to record meaningfully resolved proton spectrum of fully protonated proteins. Fast MAS ( 100 kHz) combined with high static ﬁelds has enabled protondetected correlation spectroscopy in fully protonated samples with suﬃcient resolution, primarily for the purpose of assignment.
Here, we propose a new experimental method to obtain long-range proton-proton contacts in fully protonated proteins at fast MAS. The novel method demonstrates that 1 H-1 H contacts on the order of 6-7 ˚can be obtained in fully protonated proteins. A systematic comparison of the A experimental 1H-1H contacts was performed with the expected 1H-1H contacts at a distance of 7 ˚based on the X-ray structure A