The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for alpha-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [(15)N], [(15)N, (13)C], [(15)N, (13)C, (2)H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three alpha-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg(58) site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 +/- 0.10 A, 0.40 +/- 0.13 A, and 0.57 +/- 0.19 A, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G(56)VRSG(60) region. (15)N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor.
The structure and dynamics of a large segment of Ste2p, the G-protein-coupled alpha-factor receptor from yeast, were studied in dodecylphosphocholine (DPC) micelles using solution NMR spectroscopy. We investigated the 73-residue peptide EL3-TM7-CT40 consisting of the third extracellular loop 3 (EL3), the seventh transmembrane helix (TM7), and 40 residues from the cytosolic C-terminal domain (CT40). The structure reveals the presence of an alpha-helix in the segment encompassing residues 10-30, which is perturbed around the internal Pro-24 residue. Root mean-square deviation values of individually superimposed helical segments 10-20 and 25-30 were 0.91 +/- 0.33 A and 0.76 +/- 0.37 A, respectively. 15N-relaxation and residual dipolar coupling data support a rather stable fold for the TM7 part of EL3-TM7-CT40, whereas the EL3 and CT40 segments are more flexible. Spin-label data indicate that the TM7 helix integrates into DPC micelles but is flexible around the internal Pro-24 site, exposing residues 22-26 to solution and reveal a second site of interaction with the micelle within a region comprising residues 43-58, which forms part of a less well-defined nascent helix. These findings are discussed in light of previous studies in organic-aqueous solvent systems.
Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 2 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG)[1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol(TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.
The minimal model system to study the basic principles of protein folding is the hairpin. The formation of beta-hairpins, which are the basic components of antiparallel beta-sheets, has been studied extensively in the past decade, but much less is known about helical hairpins. Here, we probe hairpin formation between a polyproline type-II helix and an alpha-helix as present in the natural miniprotein peptide YY (PYY). Both turn sequence and interactions of aromatic side chains from the C-terminal alpha-helix with the pockets formed by N-terminal Pro residues are shown by site-directed mutagenesis and solution NMR spectroscopy in different solvent systems to be important determinants of backbone dynamics and hairpin stability, suggesting a close analogy with some beta-hairpin structures. It is shown that multiple relatively weak contacts between the helices are necessary for the formation of the helical hairpin studied here, whereas the type-I beta-turn acts like a hinge, which through certain single amino acid substitutions is destabilized such that hairpin formation is completely abolished. Denaturation and renaturation of tertiary structure by temperature or cosolvents were probed by measuring changes of chemical shifts. Folding of PYY is both reversible and cooperative as inferred from the sigmoidal denaturation curves displayed by residues at the interface of the helical hairpin. Such miniproteins thus feature an important hallmark of globular proteins and should provide a convenient system to study basic aspects of helical hairpin folding that are complementary to those derived from studies of beta-hairpins.
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