My remembrances of H.G. Khorana: exploring the mechanism of bacteriorhodopsin with site-directed mutagenesis and FTIR difference spectroscopy

Abstract

H.G. Khorana's seminal contributions to molecular biology are well-known. He also had a lesser known but still major influence on current application of advanced vibrational spectroscopic techniques such as FTIR difference spectroscopy to explore the mechanism of bacteriorhodopsin and other integral membrane proteins. In this review, I provide a personal perspective of my collaborative research and interactions with Gobind, from 1982 to 1995 when our groups published over 25 papers together which resulted in an early picture of key features of the bacteriorhodopsin proton pump mechanism. Much of this early work served as a blueprint for subsequent advances based on combining protein bioengineering and vibrational spectroscopic techniques to study integral membrane proteins.

Keywords: Bacteriorhodopsin (BR); FTIR difference spectroscopy; Gobind Khorana; Integral membrane proteins; Raman spectroscopy; Rhodopsin.

Fig. 1

The BR Photocycle: After absorption of a photon (yellow arrow) by light-adapt BR₅₇₀, the photocycle consists of a series of intermediate states with different characteristic life-times that ultimately results in the returns back to the original light-adapted BR₅₇₀ state. Subscripts designate the wavelength of maximum visible absorption of each intermediate in the photocycle. Centeral image shows the color of the BR and M states. A key goal of our collaboration with the H.G. Khorana group was to elucidate conformational changes occurring in BR during each step in this photocycle (from reference Rothschild (2016))

Fig. 2

The first FTIR difference spectrum recorded of an integral membrane protein. While the FTIR absorption spectrum of purple membrane reveals little about the mechanism of BR proton transport (top trace), the difference spectrum is rich in information about protein and chromophore groups that undergo a structural change during the BR to M phototransition (bottom trace). The band at 1762 cm.⁻¹ was associated with a protonation of an Asp or Glu residue (red box), whereas other bands were associated with chromophore groups (red box) based on comparison to resonance Raman spectra (middle trace) (adapted from reference Rothschild et al. (1981))

Fig. 3

Comparison of FTIR difference spectra for WT and Asp → Asn, Glu substations in the BR amino acid sequence for the BR → K, L and M transitions recorded at low-temperature. Additional low-temperature studies on the BR → N (Bousche, Braiman et al. 1991) and BR → O transitions (Bousche et al. 1992), as well as time-resolved FTIR difference measurements (Braiman, Ahl et al. , , Braiman, Bousche et al. , , Bousche et al. 1992), led to an early model of the BR proton pump mechanism (adapted from Braiman et al. (1988a, b))