Elastin-Like Peptide as a Model for Disordered Proteins: Diffusion Behaviour in Self-Crowding Conditions.

Despite the current high interest, there is limited information on diffusion data for intrinsically disordered proteins (IDPs). This study investigates the effect of crowding on the diffusion behaviour of an elastin-like peptide (ELP), by combined pulse field gradient (PFG) and static field gradient (SFG) NMR techniques. We interpret our findings in terms of highly dynamic chain assemblies with weak interactions, resulting in ELP diffusion that is primarily governed by the viscous flow of the solvent. The diffusion behaviour of the peptide appears to resemble that of globular proteins rather than flexible linear polymers over a wide concentration range.

Detection and Verification of a Key Intermediate in an Enantioselective Peptide Catalyzed Acylation Reaction.

Until now, the intermediate responsible for the acyl transfer of a highly enantioselective tetrapeptide organocatalyst for the kinetic resolution of trans-cycloalkane-1,2-diols has never been directly observed. It was proposed computationally that a π-methylhistidine moiety is acylated as an intermediate step in the catalytic cycle. In this study we set out to investigate whether we can detect and characterize this key intermediate using NMR-spectroscopy and mass spectrometry. Different mass spectrometric experiments using a nano-ElectroSpray Ionization (ESI) source and tandem MS-techniques allowed the identification of tetrapeptide acylium ions using different acylation reagents. The complexes of trans-cyclohexane-1,2-diols with the tetrapeptide were also detected. Additionally, we were able to detect acylated tetrapeptides in solution using NMR-spectroscopy and monitor the acetylation reaction of a trans-cyclohexane-1,2-diol. These findings are important steps towards the understanding of this highly enantioselective organocatalyst.

Triplet state solvation dynamics: extending the accessible timescale by using indole as local probe.

Triplet state solvation dynamics (TSD) is a truly local measurement technique, where a dye molecule is dissolved as a probe at low concentration in a solvent. Depending on the dye molecule, local information on mechanical or dielectric solvation can be obtained. So far, this method has mainly been used to investigate topics such as fundamentals of glassy dynamics and confinement effects. Based on the procedure presented in [P. Weigl et al., Z. Phys. Chem., 2018, 232, 1017-1039] in the present contribution two new TSD probes, namely indole and its derivative cbz-tryptophan, are identified and characterized in detail. In particular, their longer phosphorescence lifetime allows for a significant extension of the timescale of local mechanical and dipolar solvation measurements. In combination with previously used dyes a measurement window of up to five orders of magnitude in time can be covered. Furthermore, we show that in cbz-tryptophan the indole unit is the phosphorescence center, while the rest of the molecule only slightly contributes to the solvation response function. The detailed understanding of these two new TSD probes presented in this work, will allow in depth investigations of solvation and the corresponding dynamics also for biologically relevant systems in the future.

Conformational Analysis of an Antibacterial Cyclodepsipeptide Active against Mycobacterium tuberculosis by a Combined ROE and RDC Analysis.

Griselimycin (GM) and methylgriselimycin (MGM), naturally produced by microorganisms of the genus Streptomyces, are cyclic depsipeptides composed of ten amino acids. They exhibit antibacterial activity against Mycobacterium species by inhibiting the sliding clamp of prokaryotic DNA polymerase III and are therefore considered as potential anti-tuberculosis drugs. The difference between the peptides is the presence of l-(R)-4-methyl-proline in MGM instead of l-proline in GM at position 8 of the amino acid sequence. Methylation increases both metabolic stability and activity of MGM compared to GM. To get deeper insight into the structure-activity relationship, the solution structure of the cyclic part of MGM was determined using rotating-frame nuclear Overhauser effect (ROE) distance restraints and residual dipolar couplings (RDC). The structure of MGM in solution is compared to the structure of GM in a co-crystal with DNA polymerase III subunit beta. As a result, a highly defined structural model of MGM is obtained, which shows related characteristics to the bound GM.

Nanoscale Biodegradable Organic-Inorganic Hybrids for Efficient Cell Penetration and Drug Delivery.

We report a comprehensive study on novel, highly efficient, and biodegradable hybrid molecular transporters. To this end, we designed a series of cell-penetrating, cube-octameric silsesquioxanes (COSS), and investigated cellular uptake by confocal microscopy and flow cytometry. A COSS with dense spatial arrangement of guanidinium groups displayed fast uptake kinetics and cell permeation at nanomolar concentrations in living HeLa cells. Efficient uptake was also observed in bacteria, yeasts, and archaea. The COSS-based carrier was significantly more potent than cell-penetrating peptides (CPPs) and displayed low toxicity. It efficiently delivered a covalently attached cytotoxic drug, doxorubicin, to living tumor cells. As the uptake of fluorescently labeled carrier remained in the presence of serum, the system could be considered particularly attractive for the in vivo delivery of therapeutics.

Synthesis of azobenzenealkylmaleimide probes to photocontrol the enzyme activity of a bacterial histone deacetylase-like amidohydrolase.

A series of azobenzenealkylmaleimides (AMDs) with different spacer length was synthesized and coupled via Michael-Addition to a specific mutant of a bacterial histone deacetylase-like amidohydrolase (HDAH). Michaelis-Menten parameters (Vmax and Km) were employed to characterize the effect of both, the spacer length and the configuration (cis vs. trans) of the attached azobenzene moiety, on the HDAH enzyme activity. The photoswitch behavior of the AMD/enzyme conjugate activity was clearly influenced by the AMD spacer length. This study highlights the importance of steric rearrangement of the photoswitch with respect to the active site and describes a strategy to optimize the photocontrol of HDAH.

Triplet States Reveal Slow Local Dynamics in the Solvation Shell of Biomolecules.

Protein hydration shell dynamics plays a pivotal role in biochemical processes such as protein folding, enzyme function, molecular recognition and interaction with biological membranes. Thus, it is crucial to understand the mobility of the solvation shell at the surface of biomolecules. Triplet state solvation dynamics can reveal the slow dynamics of the solvation shell. This is done in the present work without adding separate dye molecules but instead by using a phosphorescent subgroup of the biomolecule itself. In particular, we study a small heptapeptide in a glycerol-water mixture under cryoconservation conditions so that the system can be supercooled without crystallization. We find a significant slowing of molecules in the solvation shell in the millisecond range compared to the bulk. This opens up the possibility to unravel the nature of relaxation processes in the solvation shell usually overlapping at room temperature.