FTIR:  Fourier-Transform Infrared Spectroscopy

Summary. 
Fourier-transform infrared (FTIR) spectroscopy can be used to measure the vibrational spectrum of a complex biological macromolecule.  The Sakmar Laboratory and its collaborators, most notably Prof. Friedrich Siebert, Albert-Ludwigs-Universität, Freiburg, Germany, developed an approach to study recombinant engineered visual pigments using FTIR-difference spectroscopy.  Over a period of nearly a decade, using a combination of site-directed mutagenesis, biochemical characterization and FTIR-difference spectroscopy, members of the Sakmar Laboratory were able to assign specific IR vibrational difference bands to specific amino acid side chains in rhodopsin and to show how the bands shifted upon receptor activation.  In ongoing studies of rhodopsin structure and activity, the same approach is being combined with UAAM in collaboration with Prof. Reiner Vogel, Albert-Ludwigs-Universität.  In a recent systems biology application of FTIR spectroscopy, Dr. Thomas Huber invented an FTIR apparatus to create and measure concentration gradients of volatile odorants in air and simultaneously record the chemotactic behavior of genetically altered fruit fly larvae.

Structural Studies of Rhodopsin. 
FTIR-difference spectroscopy is a powerful method to study the structural changes that occur in rhodopsin, a prototypical G protein-coupled receptor (GPCR), as it undergoes characteristic conformational changes after photoisomerization of it 11-cis-retinal chromophore.  In the mid-1990s, the Sakmar Laboratory pioneered the use of FTIR spectroscopy studies of engineered recombinant rhodopsin expressed in mammalian cells in culture.  Specific vibrational bands, most notably the carbonyl stretching frequencies of protonated carboxylic acid residues, were assigned to specific amino acid side chains in the receptor.   The power of FTIR-difference spectroscopy is that structural changes can be followed over time using thermal trapping methods to freeze specific protein conformations while spectra are measured.  The interpretation of ongoing FTIR data has been facilitated by recent high-resolution crystal structures of rhodopsin and other related GPCRs.  FTIR studies of rhodopsin and rhodopsin mutants have contributed to validating and understanding the “helix movement model of receptor activation” and the concept of “functional microdomains” in the super family of GPCRs.

Recent studies in the Sakmar Laboratory combine FTIR-difference spectroscopy with UAA mutagenesis, in which the useful IR probe azido-phenylalanine (azF) is introduced at specific sites in rhodopsin.  The antisymmetric stretching vibration of azido results in a strong absorption band at around 2,100 wavenumbers that is distinct from other vibrational absorption bands typically present in proteins.  The strength and position of the azido band is sensitive to subtle changes of the electric field of its local environment.  In proof-of-concept studies, azF was introduced at several sites in rhodopsin.  The rhodopsin mutants were then studied with FTIR-difference spectroscopy and the results were interpreted in the context of recent structural studies with the aid of computer molecular dynamics modeling.  

Olfactory Chemotactic Behavior. 
Chemotaxis refers to directed movement toward attractive chemicals and away from aversive chemicals.  The process of chemotaxis requires continuous computation and integration of local odorant gradients and an appropriate response.  How animals detect and integrate olfactory stimuli in three-dimensional space remains a mystery.  Part of the problem in studying chemotactic behavior is that it is difficult to generate, control and measure odorant gradient in space.  The Sakmar Laboratory recently developed a new behavioral assay where the quantity and distribution of an olfactory stimulus can be controlled and precisely quantified in space.  Using the new assay, which relies on FTIR spectroscopy, we delineated the behavior and neural mechanisms underlying chemosensory orientation in genetically modified fruit fly larvae. This latest work was carried out in collaboration with Prof. Leslie Vosshall, Rockefeller University and Prof. Mathieu Louis, Centre for Genomic Regulation, Barcelona, Spain.