YIA-I Structure and Dynamics of a Ribosome-bound Nascent Chain by NMR
Shang-Te D. Hsu, Paola Fucini, Lisa D. Cabrita, Hélène Launay, Christopher M. Dobson,
YIA-II Avalanching Amplification of Sensitivity and Contrast by Feedback-Enhanced
NMR and MRI
Susie Y. Huang, Dennis W. Hwang, Jamie D. Walls, Lian-Pin Hwang, Yung-Ya Lin
YIA-III How do fungi make raincoats?
Ann Kwan, Margie Sunde, Matt Templeton, Joel Mackay
YIA-IV Supramolecular chemistry in a microfluidic NMR-chip
Aldrik Velders , Victoria Gomez
Structure and Dynamics of a Ribosome-bound Nascent Chain by NMR Spectroscopy
1 2 1 1 1
Shang-Te D. Hsu , Paola Fucini , Lisa D. Cabrita , Hélène Launay , Christopher M. Dobson , John
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United
Kingdom and Max Planck Institute for Molecular Genetics, Ihnestrasse 73, Berlin D-14196, Germany
Protein folding in living cells is inherently coupled to protein synthesis and chain elongation.
There is considerable evidence that some nascent chains fold into their native structures in a
co-translational manner prior to release from the ribosome but despite its importance a detailed
description of such a process at the atomic level remains elusive. We show that by generating
translation-arrested ribosomes in which the newly synthesised polypeptide chain is selectively
C/15N-labelled we observe, using ultrafast nuclear magnetic resonance (NMR) techniques, a
large number of resonances of a ribosome-bound nascent chain complex (RNC) corresponding
to a pair of C-terminally truncated immunoglobin (Ig) domains. Analysis of these spectra
reveals that the nascent chain adopts a structure in which a native-like N-terminal Ig domain is
tethered to the ribosome by a largely unfolded and highly flexible C-terminal domain.
Selective broadening of resonances for a group of residues that are co-localised in the structure
demonstrates that there are specific but transient interactions between the ribosome and the
N-terminal region of the folded Ig domain. These findings represent a first step towards a
detailed structural understanding of the cellular processes of co-translational folding.
Avalanching Amplification of Sensitivity and Contrast by Feedback-Enhanced NMR and MRI
1 2 3 1,4 3*
Susie Y. Huang , Dennis W. Hwang , Jamie D. Walls , Lian-Pin Hwang , Yung-Ya Lin
Harvard Medical School, Boston, MA, USA Department of Chemistry, National Taiwan University,
Department of Chemistry & Biochemistry, University of California, Los Angeles, USA, 4IAMS, Academia
Improving sensitivity and contrast is particularly important in applications of magnetic
resonance (MR) to biomolecular structure determination, where insensitive and/or dilute spins
are detected, and medical imaging, where spatial-resolving contrast is crucial to the early
diagnosis of disease. We present a new approach to MR sensitivity and contrast enhancement
that manipulates the intrinsic spin dynamics in the presence of nonlinear feedback interactions.
To enhance the dependence of the magnetization on specific MR properties, we employ a
feedback field that depends explicitly on the magnetization itself, rendering the Bloch
equations nonlinear. Sensitivity and contrast enhancement are triggered by the smallest
changes in the initial input magnetization or magnetization distribution and build up rapidly
through positive feedback to reflect the underlying MR parameters.
By manipulating bulk solvent spins near the threshold of instability to detect dilute solute
spins, sensitivity and signal-to-noise ratios (SNR) in spectroscopic measurements can be
enhanced by more than 10 times. In imaging, feedback-based MRI yields robust image
contrast that is sensitive to small differences in the underlying microscopic frequency
distributions. Important applications of this method include improving the visualization of SPIO
nanoparticles through generation of positive contrast and distinguishing small changes in
microscopic susceptibility corresponding to tumor and normal tissue. Using an external
electronic device can significantly enhance the feedback field and open opportunities for the
design of novel imaging pulse sequences in which the feedback interaction is controllable.
Examples of in vitro and in vivo tumor detection in human brain tissue and mouse models of
lung adenocarcinoma with active feedback will be demonstrated. Feedback-enhanced NMR
and MRI offer a conceptually new approach to practicing MR, in which the spins themselves
play an active role in determining and differentiating their subsequent evolution, leading to
avalanching amplification of sensitivity and contrast.
How do fungi make raincoats?
Ann Kwan1, Margie Sunde1, Matt Templeton2 and Joel Mackay1
School of MMB, University of Sydney, NSW 2006 2The Horticultural and Food Research Institute of New
Zealand, Mt Albert Research Centre, New Zealand
Many fungi produce proteins that confer water resistance to their surface structures such as
spores. EAS is a class I hydrophobin protein from the fungus Neurospora crassa and belongs
to a family of small cysteine-rich proteins that have the remarkable ability to self-assemble into
polymeric, amphipathic film on the surface of aerial structures. The hydrophobin monolayers
are extremely resistant to degradation and as such offer the possibility of many
biotechnological applications such as the reversal of surface polarity.
We have determined the solution structure of EAS monomer using multidimensional NMR
spectroscopy. The structure consists of flexible regions as well as a core beta-barrel structure,
which is stabilized by four intramolecular disulfide bonds. Interestingly, EAS displays a
complete segregation of charged and hydrophobic residues on its surface, consistent with its
ability to form an amphipathic polymer. The structure suggests the pre-formed core may allow
and guide the extremely rapid polymerisation of the hydrophobins.
To further explore the structural and functional significance of the flexible loops and the
charged and hydrophobic patches in hydrophobin assembly, we have set up an expression
protocol for EAS in bacteria. We are currently using mutagenesis, NMR spectroscopy, X-ray
fiber diffraction and electron microscopy studies to probe the effect of loop sequence on
hydrophobin film morphology and function. So far, we have shown a large part of the flexible
loop can be removed from EAS without affecting its structure or its ability to self-polymerise.
We have also identified key regions that are important for the hydrophobin self-assembly
process. Our study will provide molecular insight into the structure and assembly process of
Supramolecular chemistry in a microfluidic NMR-chip
Victoria Gomez, Aldrik Velders*
Supramolecular Chemistry & Technology, MESA+ Institute for Nanotechnology, University of Twente,
Enschede, The Netherlands.
In this presentation we will show the potential of a microfluidic chip equipped with a planar
transceiver microcoil (viz. an NMR-chip) to investigate supramolecular structures and
host-guest interactions in nanoLiter sample volumes. Proof-of-principle is obtained
characterizing picomol quantities of supramolecular ‘host’ systems and quantifying their
interactions with different ‘guest’ molecules. Initial work on microliter-volume NMR probes has
focused on solenoid microcoil designs, because of their higher sensitivity compared to planar
microcoils. However, the latter ones are more easily to integrate in microfabrication processes
using photolithographic techniques, allowing a precisely controlled geometry and high degree
of flexibility in the design of the microfluidic channels and coil parameters. The latest
developments regarding design and fabrication of our NMR-chips with an active volume of less
than 10 nL will be presented, showing a surprisingly good performance at 9.4 T (400 MHz)
systems. The high resolution and high sensitivity obtained with the new generation NMR-chips
offer the possibility to work with more complicated chemical structures and at relatively low
concentrations, opening up the window to study for example supramolecular assemblies inside
the NMR-chip. The double-rosette motif is a rather complicated self-assembled network of
hydrogen bonds formed between three melamines and three barbituric acids forming
thermodynamically stable molecular “boxes”. 1D and 2D 1H-NMR experiments run in our
NMR-chip show on the one hand that it is possible to identify this kind of supramolecular
assemblies in picomol amounts (nanogram range). On the other hand, titration experiments
can readily be performed in the NMR-chip observing shifts in the host protons resonances
when different equivalents of guests, e.g. 4-nitrophenol, are added. In conclusion, it is the first
time that supramolecular assemblies and the concomitant host-guest chemistry are
investigated in an NMR-chip.