Centrality of Weak Interhelical H-bonds in Membrane Protein Functional Assembly and Conformational Gating

Our analysis demonstrates that backbone-mediated interhelical hydrogen-bonds cluster laterally in the conserved core of transmembrane helical proteins. Each residue's propensity to bear these interactions is in correlation with the residue's packing-value scale; giving biophysical meaning to this phenomenological scale. Residues participating in such an intersubunit, structurally conserved H-bond in reaction centers of photosystem II were combinatorially mutated and characterized in silico and in vivo suggesting that Hbond reversible association regulates protein-gated electron transfer. Similar motifs may be involved in folding and conformational flexibility of other membrane proteins. Hence, these findings provide new parameters for structure and function prediction. Contact: ilan.samish@weizmann.ac. il INTRODUCTION Membrane protein conformational gating plays key roles in protein systems (Spencer and Rees 2002). Although functionally distinct in many catalytic cycles, the underlying mechanisms remain enigmatic (Warshel and Parson 2001). In order to flip between two alternative conformations, weak interactions must rearrange. Such interactions may include weak (as defined by Desiraju (Desiraju and Steiner 1999)) and strong interhelical H-bonds as well as packing interactions (Richards 1974). Examples to weak bonds we studied include: (a) A bifurcated H-bond in which a backbone carbonyl accepts an interand intrahelical Hbond. (b) An interhelical bond donated by the acidic Cα atom (Senes, Ubarretxena-Belandia et al. 2001). Amplified by the low dielectric milieu of the membrane, weak interactions in membrane proteins have a stronger effect in comparison to their soluble counterparts. More generally, similar interactions are involved in membrane protein functional assembly (DeGrado, Gratkowski et al. 2003) playing a part in the helix-assembly step within the 3-stage model for membrane protein folding (Engelman, Chen et al. 2003). Following statistical characterization of interhelical Hbonds in a non-redundant set of structurally known helical proteins, we concentrated on photosynthetic reaction centers (PRCs) as a model system – the 1 and most varied group of structurally-known helical membrane proteins. Due to 3.5 billion years of evolution, these protein complexes have undergone considerable sequence degeneracy, but have structurally maintained important motifs required for function. Consequently, functionally important and structurally conserved motifs can be datamined followed by discrete analysis of the common motif in each available structure. Further, the study of membrane proteins in their natural membrane environment imposes technical difficulties due to the lack of ‘reporting agents’ that can teach us about molecular level changes. Hence, the multistep electron transfer (ET) in PRCs can serve as a molecular reporter to changes in different microenvironments of the protein. Last, photosystem II PRC in cyanobacteria is a wellsuited system for genetic manipulation and in vivo biophysical characterization. Thus, combinatorial mutagenesis, combined with temperature-dependent biophysical analysis and in silico mutant characterization enables one to utilize PRCs as a model system to study the role/fitness of different residues/chemical moieties in the functional assembly and conformational gating of membrane proteins. Our study integrates bioinformatic tools in a feedback loop – guiding the genetic manipulation strategy and explaining the resulting biophysical findings. Generalization of key findings in the model system is presented by searching for the phenomenon in sequence and structure space and vice versa. Finally, the recent accumulation of helical membrane protein structures enabled us to conduct statistical datamining of the phenomenon suggested by our model system in a non-redundant dataset of all helical membrane proteins. Cumulatively, our results suggest new roles for weak interhelical H-bonds in the membrane milieu as well as biophysical meaning to interactions previously regarded phenomenologically as 'packing'. RESULTS & DISCUSSION A structural core of all PRCs was computed based on the multiple structural alignment and core alignment algorithm. 200 out of 700 locations of Cα-atoms were structurally conserved in all PRCs. A cluster of high-packing amino acids in the central core of all complexes was found. This cluster was analyzed in theoretical and crystal structures of type II (i.e. bacterial and photosystem II) PRCs. Conserved