Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change / Soumit Sankar Mandal ... [et al.]. In: Proceedings of the National Academy of Sciences of United States of America. - ISSN 0027-8424. - Vol. 114, no. 23 (2017), s. 6040-6045. [MANDAL, Soumit Sankar (20%) - MERZ, Dale R. (20%) - BUCHSTEINER, Maximilian (5%) - DIMA, Ruxandra I. (5%) - RIEF, Matthias (5%) - ŽOLDÁK, Gabriel (45%) ]

Analysis of IgG kinetic stability by differential scanning calorimetry, probe fluorescence and light scattering / Michal Nemergut ... [et al.]. - Č. projektu: VVGS 2016-72642,, VEGA 1/0423/16, APVV 15-0069. In: Protein Science. - ISSN 0961-8368. - Vol. 26, no. 11 (2017), s. 2229-2239. [NEMERGUT, Michal (15%) - ŽOLDÁK, Gabriel (10%) - SCHAEFER, Jonas V. (5%) - KAST, Florian (5%) - MIŠKOVSKÝ, Pavol (5%) - PLÜCKTHUN, Andreas (10%) - SEDLÁK, Erik (50%) ]

Subnanometre enzyme mechanics probed by single-molecule force spectroscopy / Benjamin Pelz ... [et al.]. In: Nature Communications. - ISSN 2041-1723. - Vol. 7 (2016), s. 10848-10848. [PELZ, Benjamin (40%) - ŽOLDÁK, Gabriel (20%) - ZELLER, Fabian (10%) - ZACHARIAS, Martin (10%) - RIEF, Matthias (20%) ]

Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK / Daniela Bauer ... [et al.]. In: Proceedings of the National Academy of Sciences of the United States of America. - ISSN 0027-8424. - Vol. 112, no. 33 (2015), p. 10389-10394. [BAUER, Daniela (20%) - MERZ, Dale R. (5%) - PELZ, Benjamin (5%) - THEISEN, Kelly E (5%) - YACYSHYN, Gail (5%) - MOKRANJAC, Dejana (5%) - DIMA, Ruxandra I. (5%) - RIEF, Matthias (20%) - ŽOLDÁK, Gabriel (20%) ]

A Compact Native 24-Residue Supersecondary Structure Derived from the Villin Headpiece Subdomain / Henry G. Hocking ... [et al.]. In: Biophysical Journal. - ISSN 0006-3495. - Vol. 108, no. 3 (2015), s. 678-686. [HOCKING, Henry G. (20%) - HAESE, Florian (20%) - MADL, Tobias (5%) - ZACHARIAS, Martin (5%) - RIEF, Matthias (5%) - ŽOLDÁK, Gabriel (45%) ]

01/2019 - 12/2021, Principal investigator, VEGA 1/0175/19, Aggregation of immunoglobulins and prediction of their colloidal stability using advanced kinetic analyzes,

The stability of immunoglobulins G (IgG) is one of the key features influencing successful clinical application immunotherapeutics. Obtaining correct parameters describing IgG stability and aggregation is an ambitious task mainly due to the complexity of the multi-domain structure of IgG. Our group has developed a conformational kinetic model stability, which can determine the kinetic and conformational stability of the protein based on the analysis of IgG temperature profiles over a wide temperature range. In this project we find both exogenous (eg pH, ionic strength, osmolytes) and endogenous (e.g. glycosylation, mutations, fragmentation) factors affect the kinetic and colloidal stability of selected IgGs. Our methodologies developed in previous projects will allow us to understand the molecular nature mentioned factors. The obtained experimental data will further allow us to obtain a unique set of parameters kinetic stability, which we will be able to implement in the conceptual development of predictive methods and procedures aimed at determining the colloidal stability of IgG. 

01/2019 - 12/2023, Principal investigator, APVV-18-0285, Understanding the mechanism of IgG inactivation using individual molecules of Hsp70 shaperone and laser optical tweezers,

Owing to their clinically proved therapeutic efficacy, antibodies of the IgG class are the most attractive group of proteins for research and development. However, their structural and biochemical complexity, as well as unclear inactivation mechanism, provide a significant hurdle for their accelerated development and their use in the clinical applications. In particular, the major concern during early stages of research and development is a natural tendency of antibodies to lose their efficacy by unproductive protein-protein association, which is likely

triggered by the local unfolding of certain critical parts of the IgG structure. These parts expose sticky hydrophobic parts, which interact with multiple IgG and lead to supramolecular structures of inactive antibodies. Hence, it is a great challenge to understand and suppress aggregation-prone states; however, due to their transient nature, such spots are invisible to conventional experimental techniques. In fact, a general method for monitoring exposed hot spots in the antibody does not exist. To this end, in the proposed project we plan to combine single-molecule sensitivity, optical tweezers and the very fundamental property of heat-shock chaperone proteins to bind hydrophobic patches of thermally stressed proteins. Specifically, we will in-depth characterize our new single-molecule heat shock protein 70 (E.coli Hsp70) sensor

to detect low-populated states of IgG with exposed hydrophobic patches. In parallel, we will complement our study by scrutinizing the IgG inactivation using conventional studies which will be used to assess the predictive capacity of our chaperone sensor. Next, we will perform a screening of low molecular weight compound libraries to discover molecules inhibiting IgG inactivation monitored by our Hsp70

sensor.

01/2021 - 12/2023, Principal investigator, KEGA 005UPJŠ-4/2021, eduLab - educational laboratory of lab-on-a-chip technology for accelerating innovations of diagnostic methods in Slovakia,

The current pandemic situation has radically defined new specific requirements for teaching the conceptual development of diagnostic tools with the task of protecting the health of society. The development of modern diagnostic methods requires a combination of biological and theoretical-experimental physical interdisciplinary approach, which is currently absent in the academic environment. Students will gradually focus on the basic principles of microfluidic devices and their physical modeling, they will design their own lab-on-a-chip devices with the ability of laser optical manipulation of microparticles and evaluation of measured data. The tools and equipment of the laboratory will enable students to acquire practical and theoretical skills for a comprehensive mastery of modern lab-on-a-chip technologies with potential

for the development of own applications, e.g. in the field of diagnostics in biomedicine. 

01 / 2021-06 / 2023, Project coordinator, 313011AUW6, Biopickmol, Development of nanosensory photonic systems for rapid virus detection using methods of controlled evolution of protein platforms: the case of SARS-CoV-2.

Development of detection photonic system, development of specific detection chips, Functionalization of chip surface by nanolayer of specific protein molecules developed by the method of controlled evolution of selected protein platforms. The formation of protein nanolayers on the detection chip will take place by means of controlled specific chemical conjugation, thus achieving optimal binding efficiency and reproducibility of detection properties. After loading the inactivated virus sample on the functionalized chip, the presence of the virus will be detected based on the amplified Raman spectrum by surface plasmon. The bioRAMASCOPE detection system will be developed to detect the signal from the functionalized chips. bioRAMASCOPE will meet the specific requirements regarding detection limits and operational reliability required for SARS-CoV-2 virus detection. Using modern protein engineering selection methods, artificial proteins will be developed as new biological entities with a binding function that specializes in the specific binding of inactivated viruses. Based on the combination of different epitope binding, we plan to achieve high sensitivity and specificity of selected protein platforms against different types of viruses, including SARS-CoV-2.

01/2016 – 12/2017, principal investigator, PPP DAAD Tschechien 2 J.. Design of fluorescence-based analytical tool for quantification of the protein-protein interaction within the 14-3-3 family. Bilateral grant. ID. 5020923