Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II from Natronomonas pharaonis

Chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes. Previous studies of the cytoplasmic fragment of the phototaxis transducer (pHtrII-cyt) from the halophilic archaeon Natronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions, respectively. CD spectra revealed only approximately 24% helical structure, even in 4 M KCl, leaving it an open question how the rod-like shape is achieved. Here, we conducted CD, FTIR, and NMR spectroscopic studies under different conditions to address this question. We provide evidence that pHtrII-cyt is highly dynamic with strong helical propensity, which allows it to change from monomeric to dimeric helical coiled-coil states without undergoing dramatic shape changes. A statistical analysis of predicted disorder for homologous sequences suggests that structural flexibility is evolutionarily conserved within the methyl-accepting chemotaxis protein family

FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer HtrI

Mironova OS, Budyak IL, Bueldt G, Schlesinger R, Heberle J. FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer HtrI. BIOCHEMISTRY. 2007;46(33):9399-9405.The phototaxis receptor sensory rhodopsin I (SRI) from Halobacterium salinarum interacts with its cognate transducer (HtrI) forming a transmembrane complex. After light excitation of the chromophore all-trans retinal, SRI undergoes structural changes that are ultimately transmitted to HtrI. The interaction of SRI with HtrI results in the closure of the receptor's proton pathway, which renders the photocycle recovery kinetics of SRI pH-independent. We demonstrate on heterologously expressed and reconstituted SRI-HtrI fusion proteins that the transmembrane part of HtrI (residues 1-52) as well as the downstream cytoplasmic part (residues 53-147) exhibit conformational changes after light excitation. The sum of these conformational changes is similar to those observed in the fusion constructs SRI-HtrI 1-71 and SRI-HtrI 1-147, which display pH-independent receptor kinetics. These results indicate the occurrence of spatially distinct conformational changes that are required for functional signal transmission. Kinetic and spectroscopic analysis of HtrI point mutants of Asn53 provides evidence that this residue is involved in the receptor-transducer interaction. We suggest that Asn53 plays a role similar to that of Asn74 of the HtrII from Natronobacterium pharaonis, the latter forming a hydrogen bond to the receptor within the membrane

Delicate Balance between Functionally Required Flexibility and Aggregation Risk in a β‑Rich Protein

Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly β-sheet protein whose folding has been explored in detail, cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the challenge of understanding the links between a protein’s natural tendency to fold, ‘breathe’, and function with its propensity to misfold and aggregate. We identified near-native dynamic species that lead to aggregation and found that inherent structural fluctuations in the native protein, resulting in opening of the ligand-entry portal, expose hydrophobic residues on the most vulnerable aggregation-prone sequences in CRABP1. CRABP1 and related intracellullar lipid-binding proteins have not been reported to aggregate inside cells, and we speculate that the cellular concentration of their open, aggregation-prone conformations is sufficient for ligand binding but below the critical concentration for aggregation. Our finding provides an example of how nature fine-tunes a delicate balance between protein function, conformational variability, and aggregation vulnerability and implies that with the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable but controllable risk

Glutamic acid-rich proteins of rod photoreceptors are natively unfolded

The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP′ part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of “natively unfolded” proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs