Infrared spectroscopy of proteins.

When Szent-Gyorgyi called water the “matrix of life”,1 he was echoing an old sentiment. Paracelsus in the 16th century said that “water was the matrix of the world and of all its creatures.”2 But Paracelsus’s notion of a matrixsan active substance imbued with fecund, life-giving propertiess was quite different from the picture that, until very recently, molecular biologists have tended to hold of water’s role in the chemistry of life. Although acknowledging that liquid water has some unusual and important physical and chemical propertiessits potency as a solvent, its ability to form hydrogen bonds, its amphoteric naturesbiologists have regarded it essentially as the backdrop on which life’s molecular components are arrayed. It used to be common practice, for example, to perform computer simulations of biomolecules in a vacuum. Partly this was because the computational intensity of simulating a polypeptide chain was challenging even without accounting for solvent molecules too, but it also reflected the prevailing notion that water does little more than temper or moderate the basic physicochemical interactions responsible for molecular biology. What Gerstein and Levitt said 9 years ago remains true today: “When scientists publish models of biological molecules in journals, they usually draw their models in bright colors and place them against a plain, black background”.3 Curiously, this neglect of water as an active component of the cell went hand in hand with the assumption that life could not exist without it. That was basically an empirical conclusion derived from our experience of life on Earth: environments without liquid water cannot sustain life, and special strategies are needed to cope with situations in which, because of extremes of either heat or cold, the liquid is scarce.4-6 The recent confirmation that there is at least one world rich in organic molecules on which rivers and perhaps shallow seas or bogs are filled with nonaqueous fluidsthe liquid hydrocarbons of Titan7smight now bring some focus, even urgency, to the question of whether water is indeed a * E-mail: p.ball@nature.com. Philip Ball is a science writer and a consultant editor for Nature, where he worked as an editor for physical sciences for more than 10 years. He holds a Ph.D. in physics from the University of Bristol, where he worked on the statistical mechanics of phase transitions in the liquid state. His book H2O: A Biography of Water (Weidenfeld & Nicolson, 1999) was a survey of the current state of knowledge about the behavior of water in situations ranging from planetary geomorphology to cell biology. He frequently writes about aspects of water science for both the popular and the technical media.

https://www.philipball.co.uk/images/stories/docs/pdf/ChemRev_final.pdf

Water as an Active Constituent in Cell Biology

Life on earth is almost entirely solar-powered. We can get some idea of the enormous quantity of energy received from the sun by noting that during daylight hours, the sun provides several thousand times more power to the surface of the U.S.A. than is produced by all of the nation’s electrical power stations. 1,2 Around 50% of the radiation that reaches the earth’s surface, roughly the visible region, is of a frequency useful to photosynthetic organisms. Oxygenic photosynthetic organisms convert this radiation into chemical energy, in the form of carbohydrate and dioxygen, at an optimal efficiency of something like 25%. 3 These products together sustain the rest of aerobic life, with carbohydrate acting as a source of high-energy electrons and dioxygen providing a lower-energy destination for these electrons. The overall equation of oxygenic photosynthesis is given in eq 1, where (CH2O) represents carbohydrate:

Water-splitting chemistry of photosystem II.

Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases.

Organisms of all domains of life use photoreceptor proteins to sense and respond to light. The light-sensitivity of photoreceptor proteins arises from bound chromophores such as retinal in retinylidene proteins, bilin in biliproteins, and flavin in flavoproteins. Rhodopsins found in Eukaryotes, Bacteria, and Archaea consist of opsin apoproteins and a covalently linked retinal which is employed to absorb photons for energy conversion or the initiation of intra- or intercellular signaling.1 Both functions are important for organisms to survive and to adapt to the environment. While lower organisms utilize the family of microbial rhodopsins for both purposes, animals solely use a different family of rhodopsins, a specialized subset of G-protein-coupled receptors (GPCRs).1,2 Animal rhodopsins, for example, are employed in visual and nonvisual phototransduction, in the maintenance of the circadian clock and as photoisomerases.3,4 While sharing practically no sequence similarity, microbial and animal rhodopsins, also termed type-I and type-II rhodopsins, respectively, share a common architecture of seven transmembrane α-helices (TM) with the N- and C-terminus facing out- and inside of the cell, respectively (Figure ​(Figure11).1,5 Retinal is attached by a Schiff base linkage to the e-amino group of a lysine side chain in the middle of TM7 (Figures ​(Figures11 and ​and2).2). The retinal Schiff base (RSB) is protonated (RSBH+) in most cases, and changes in protonation state are integral to the signaling or transport activity of rhodopsins.



Figure 1

Topology of the retinal proteins. (A) These membrane proteins contain seven α-helices (typically denoted helix A to G in microbial opsins and TM1 to 7 in the animal opsins) spanning the lipid bilayer. The N-terminus faces the outside of the cell ...

Microbial and animal rhodopsins: structures, functions, and molecular mechanisms.

Much progress has been made in our understanding of water molecule reactions on surfaces, proton solvation in gas-phase water clusters and proton transfer through liquids. Compared with our advanced understanding of these physico-chemical systems, much less is known about individual water molecules and their cooperative behaviour in heterogeneous proteins during enzymatic reactions. Here we use time-resolved Fourier transform infrared spectroscopy (trFTIR) and in situ H2(18)O/H2(16)O exchange FTIR to determine how the membrane protein bacteriorhodopsin uses the interplay among strongly hydrogen-bonded water molecules, a water molecule with a dangling hydroxyl group and a protonated water cluster to transfer protons. The precise arrangement of water molecules in the protein matrix results in a controlled Grotthuss proton transfer, in contrast to the random proton migration that occurs in liquid water. Our findings support the emerging paradigm that intraprotein water molecules are as essential for biological functions as amino acids.

Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy

Proton transfer is crucial for many enzyme reactions. Here, we show that in addition to protonatable amino acid side chains, water networks could constitute proton-binding sites in proteins. A broad IR continuum absorbance change during the proton pumping photocycle of bacteriorhodopsin (bR) indicates most likely deprotonation of a protonated water cluster at the proton release site close to the surface. We investigate the influence of several mutations on the proton release network and the continuum change, to gain information about the location and extent of the protonated water network and to reveal the participating residues necessary for its stabilization. We identify a protonated water cluster consisting in total of one proton and about five water molecules surrounded by six side chains and three backbone groups (Tyr-57, Arg-82, Tyr-83, Glu-204, Glu-194, Ser-193, Pro-77, Tyr-79, and Thr-205). The observed perturbation of proton release by many single-residue mutations is now explained by the influence of numerous side chains on the protonated H bonded network. In situ hydrogen/deuterium exchange Fourier transform IR measurements of the bR ground state, show that the proton of the release group becomes localized on Glu-204 and Asp-204 in the ground state of the mutants E194D and E204D, respectively, even though it is delocalized in the ground state of wild-type bR. Thus, the release mechanism switches between the wild-type and mutated proteins from a delocalized to a localized proton-binding site.

/pdf/proton-binding-within-a-membrane-protein-by-a-protonated-5bv1k7rbl8.pdf

Proton binding within a membrane protein by a protonated water cluster

https://www.cell.com/biophysj/pdf/S0006-3495(04)73738-8.pdf

The Assignment of the Different Infrared Continuum Absorbance Changes Observed in the 3000-1800-cm-1 Region during the Bacteriorhodopsin Photocycle

Polarized Fourier transform infrared (FTIR) difference spectroscopy has been combined with in situ H/D exchange measurements to investigate the orientation of single protonated carboxylic acids within bacteriorhodopsin (bR), exclusively based on the protein ground state. The combination of these two techniques enables the determination of the C=O dipole moment direction of D115 and D96 relative to the membrane plane to 68 +/- 11 and 45 +/- 4 degrees , respectively. By discussing these results in the context of X-ray structure analysis, we are able to determine which of the two oxygen atoms of the respective carboxylic acids binds the proton. In the case of D115, it is the oxygen which is located close to T90. On the basis of this finding, we show a possible interaction path between D115 and D85, which has been proposed to be responsible for the inhibition of the proton pump efficiency to prevent over-acidification of the external medium known as the back-pressure effect. Because the orientation of carboxylic acids can be determined even when the group does not undergo any protonation changes during the photocycle, as shown in the case of D115, the method can be applied to any orientable protein and is not merely restricted to bR.

http://www.bph.rub.de/homepage/gerwert/107.pdf

Florian Garczarek

Papers

Functional waters in intraprotein proton transfer monitored by FTIR difference spectroscopy

Proton binding within a membrane protein by a protonated water cluster

The Assignment of the Different Infrared Continuum Absorbance Changes Observed in the 3000-1800-cm-1 Region during the Bacteriorhodopsin Photocycle

Polarized FTIR spectroscopy in conjunction with in situ H/D exchange reveals the orientation of protein internal carboxylic acids.