Probing the dynamic landscape of peptides in molecular assemblies by synergized NMR experiments and MD simulations.

Peptides or proteins containing small biomolecular aggregates, such as micelles, bicelles, droplets and nanodiscs, are pivotal in many fields ranging from structural biology to pharmaceutics. Monitoring dynamics of such systems has been limited by the lack of experimental methods that could directly detect their fast (picosecond to nanosecond) timescale dynamics. Spin relaxation times from NMR experiments are sensitive to such motions, but their interpretation for biomolecular aggregates is not straightforward. Here we show that the dynamic landscape of peptide-containing molecular assemblies can be determined by a synergistic combination of solution state NMR experiments and molecular dynamics (MD) simulations. Solution state NMR experiments are straightforward to implement without an excessive amount of sample, while direct combination of spin relaxation data to MD simulations enables interpretation of dynamic landscapes of peptides and other aggregated molecules. To demonstrate this, we interpret NMR data from transmembrane, peripheral, and tail anchored peptides embedded in micelles. Our results indicate that peptides and detergent molecules do not rotate together as a rigid body, but peptides rotate in a viscous medium composed of detergent micelle. Spin relaxation times also provide indirect information on peptide conformational ensembles. This work gives new perspectives on peptide dynamics in complex biomolecular assemblies.

Tying up the Loose Ends: A Mathematically Knotted Protein.

Knots have attracted scientists in mathematics, physics, biology, and engineering. Long flexible thin strings easily knot and tangle as experienced in our daily life. Similarly, long polymer chains inevitably tend to get trapped into knots. Little is known about their formation or function in proteins despite >1,000 knotted proteins identified in nature. However, these protein knots are not mathematical knots with their backbone polypeptide chains because of their open termini, and the presence of a "knot" depends on the algorithm used to create path closure. Furthermore, it is generally not possible to control the topology of the unfolded states of proteins, therefore making it challenging to characterize functional and physicochemical properties of knotting in any polymer. Covalently linking the amino and carboxyl termini of the deeply trefoil-knotted YibK from Pseudomonas aeruginosa allowed us to create the truly backbone knotted protein by enzymatic peptide ligation. Moreover, we produced and investigated backbone cyclized YibK without any knotted structure. Thus, we could directly probe the effect of the backbone knot and the decrease in conformational entropy on protein folding. The backbone cyclization did not perturb the native structure and its cofactor binding affinity, but it substantially increased the thermal stability and reduced the aggregation propensity. The enhanced stability of a backbone knotted YibK could be mainly originated from an increased ruggedness of its free energy landscape and the destabilization of the denatured state by backbone cyclization with little contribution from a knot structure. Despite the heterogeneity in the side-chain compositions, the chemically unfolded cyclized YibK exhibited several macroscopic physico-chemical attributes that agree with theoretical predictions derived from polymer physics.

NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample.

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% 15N-labeled and uniformly 100% [13C, 15N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% 13C, 100% 15N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [13C, 15N]-labeled sample. Our results suggest that one [20% 13C, 100% 15N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [13C, 15N]-labeling for backbone resonance assignments, NMR structure determination, 15N-relaxation analysis, and ligand-protein interaction.

NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein.

The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.