BME Semineri - Erhan Deniz (Goethe University Germany) - 5 Eylül 2019 saat:10.00 sınıf: E102
Title: Infrared labels in steady-state and transient vibrational spectroscopy: Detecting conformational changes and vibrational energy transfer in proteins
Erhan Deniz, Katharina B. Eberl, Jan G. Löffler, Georg Wille, Werner Mäntele and Jens Bredenbeck
Institute of Biophysics, Goethe University Frankfurt/Main, Germany
Time and Place: September 5, 2019 @10.00 in E102
Abstract: Infrared (IR) labels provide localized insights into protein structure and dynamics. The thiol (SH) stretching mode of cysteine absorbs around 2550 cm-1 without any other overlapping protein absorption. The precise location and shape of the SH-band depends on the thiol H-bonding environment. We employ these features to monitor structural rearrangements in proteins.
We applied redox difference spectroscopy to pyruvate oxidase from E. coli (EcPOX) – a peripheral redox membrane protein that intermittently binds to the cell membrane where it feeds the respiratory chain with electrons. We identified two cysteines (out of ten) with a distinct SH band-shift upon redox reaction. These thiols sense the redox-induced exposure of the C-terminal membrane binding anchor, even though they are quite remote from the C-terminus.
Non-canonical amino acids (ncAA) can also provide IR signatures spectrally separated from the crowded amide region. We established the azide stretching mode (~ 2100 cm-1) of azidohomoalanine (Aha) as a sensor for vibrational energy, i.e. heat. Together with azulenylalanine – an ultrafast heater – we have a protein-compatible ncAA pair in hand for studying vibrational energy transfer (VET) in peptides and proteins.
VET is an excellent reporter for mechanically coupled amino acids, which might play a key role in dynamic allostery, i.e. allosteric communication without major conformational change. We applied ultrafast pump- probe spectroscopy to tryptophan zippers – short peptides with a stable hairpin structure – and the PDZ3 domain of the PSD95, a paradigm for dynamic allosteric regulation. We observe VET on a picosecond timescale along covalent bonds and non- covalent contacts, e.g. H-bonds. VET timing nicely correlates with the VET pair distance and can currently be resolved up to a distance of 17 Å.