Elucidating the function of newly identified small proteins in Methanosarcina mazei

• Applicant: Professorin Dr. Ruth Anne Schmitz-Streit
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 559991210

In all three domains of life, small proteins with less than 71 amino acids in length play important roles in diverse cellular processes such as metabolism, stress response or regulation of transcription and translation. They have been overlooked for decades due to methodological reasons and are thus often missing in genome annotations. Novel detection methods like Ribo-Seq and mass spectrometry optimized for small proteins opened up a new field of interest and catalogs of small proteins in bacteria and archaea have been recently reported. However, only a small proportion of the newly identified small proteins is characterized. The proposal aims to fill this research gap by investigating the function of selected small proteins in the methanoarchaeon Methanosarcina mazei. Our previous work identified a high number of novel small sORFs and the respective small proteins in M. mazei, of which several ones have been initially studied by biochemical and genetic approaches. The obtained results strongly argue for important physiological functions of several small proteins in biological processes in M. mazei. Consequently, we now aim to elucidate and study in detail the function(s) and corresponding molecular mechanisms of selected verified small proteins using biochemical and genetic approaches. (i) We will study the AmtB1-sP36-GlnK1 complexes on the molecular and structural level including cryo-EM analysis and gain insight into the complex composition and structure as well as into the molecular mechanism of AmtB1 inhibition by the small protein sP36 and GlnK1. (ii) sORF05 organized in an operon with two components of the trimethylamine (TMA) transporter will be studied aiming to evaluate its function in the TMA transport on the molecular level. (iii) The complex formation of GlnA1 and GOGAT and the predicted function of small protein sP26 and GlnK1 in this complex formation will be studied by different approaches including mass photometry and cryo-EM. (iv) The involvement and function(s) of the newly identified small zinc finger proteins in translation initiation or as part of the ribosome will be elucidated using different biochemical approaches including cryo-EM and crystallography as well as genetic approaches. Overall, we are expecting to gain a deep insight into the function and molecular regulatory mechanism of the selected verified small proteins involved in protein complex formation, transport processes and translation in M. mazei. Particularly elucidating the function of the newly identified small zinc finger proteins in translation initiation will open up a new field regarding translation initiation in M. mazei and allow first insights into ribosomes of Methanoarchaea.

• DFG Programme: Research Grants