Nucleic Acids Research

Orlando de Lange, Christina Wolf, Philipp Thiel, Jens Krüger, Christian Kleusch,Oliver Kohlbacher, Thomas Lahaye

Abstract
Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.

Keywords
Monolith NT.115, Protein-Nucleic Acids, Biomolecular Interaction, Prometheus NT.48, Protein Stability

In society, failure in communication can lead to conflicts in many ways. Similarly, within an organism context, failure in communication between and within cells can cause many diseases. Among the mechanisms to establish precise cellular communication is ubiquitination, a post-translational modification with the highly conserved 76-residue small protein ubiquitin. A classic example of signaling through this ubiquitin 'tag' is the targeting of obsolete proteins for degradation in the proteosome. But it also plays important roles in regulating the immune system, where it controls transcription factors that orchestrate immune resposes.

In August 25 issue of Cell, the cause of a potentially fatal inherited autoimmune disease was identified for the first time involving a dysfunction of the ubiquitin system. In this disease, now named OTULIN-related autoinflammatory syndrome (ORAS), the immune system of patients spontaneously activates and start attacking the patient's own body shortly after birth, eventually leading to the child's death. The researchers of this study showed that the disease is caused by the simple substitution of a Leucine for a Proline in the deubiquitinating enzyme OTULIN, which acts as a brake preventing the immune system to take off without permission. Without this enzyme, the levels of linear ubiquitin is uncontrolled leading to a hyperactivated immune response even in the absence of infection.

In this work, nanoDSF played a crucial role in the analysis and comparison of stability and activity between normal and disease-causing mutated OTULIN. Moreover, this breakthrough discovered a treatment for ORAS, with an antibody that blocks TNF, one of the primary molecules that mediate the activation of the immune system. Luckily this antibody drug (Infliximab or Remicade) is already approved for the treatment of other autoinmune and inflammatory diseases making it easily accesible to patients and a repurposing of drugs.

We are proud to see that nanoDSF was applied in this great study.

We care about your research

Methods in Enzymology

Deniz B. Temel, Pavel Landsman, Mark L. Brader

Abstract
Evaluating prospective protein pharmaceutical stability from accelerated screening is a critical challenge in biotherapeutic discovery and development. Measurements of protein unfolding transitions are widely employed for comparing candidate molecules and formulations; however, the interrelationships between intrinsic protein conformational stability and pharmaceutical robustness are complex and thermal unfolding measurements can be misleading. Beyond the discovery phase of drug development, astute formulation design is one of the most crucial factors enabling the protein to resist damage to its higher order structure-initially from bioprocessing stresses, then from stresses encountered during its journey from the product manufacturing site to the bloodstream of the patient. Therapeutic monoclonal antibodies are multidomain proteins that represent a large and growing segment of the biotechnology pipeline. In this chapter, we describe how differential scanning calorimetry may be leveraged synergistically with isothermal chemical denaturation and intrinsic fluorescence with concomitant static light scattering to elucidate characteristics of mAb unfolding and aggregation that are helpful toward understanding and designing optimal pharmaceutical compositions for these molecules.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Crispin G. Alexander, Randy Wanner, Christopher M. Johnson, Dennis Breitsprecher, Gerhard Winter, Stefan Duhr, Philipp Baaske, Neil Ferguson

Abstract
Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein-ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide-PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes.

Keywords 
nanoDSF, Chemical denaturation, Thermal denaturation

The human immunodeficiency virus (HIV) is responsible for the acquired immune deficiency syndrome (AIDS). While there is currently no cure for HIV/AIDS, combinations of drugs are currently used as treatment to control the viral spread either by blocking its entrance to cells or by prohibiting the virus from making copies of itself. However, a study published in the May 30 issue of Nature, took a closer look to the structure of HIV's protein shell, also known as the capsid, revealing previously unknown structural and functional features that could be targeted as a powerful alternative to current HIV therapies.

With the combination of atomic-level structural work, virology and nanoDSF, new structural details revealed that there are strongly positively charged pores in the capsid, through which nucleotides are imported at great speed to allow the viral genome to be made. These size selective pores open and close keeping out any unwanted molecules, explaining why HIV is so successful at evading the immune system.

Moreover, it was shown that by blocking the capsid pore by a small molecule, HIV reverse transcription was inhibited, leaving the HI virus unable to copy itself and thus becoming non-infectious.

This capsid feature may be common to other viruses and will be an attractive target for new antiviral drugs.

We are excited that nanoDSF played an important role in this discovery!

We care about your research