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We report high resolution solution 19 F NMR spectra of fluorinelabeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF3-CH2-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6 -10 mg), in dodecylmaltoside, were analyzed at 20°C by solution 19 F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for 19 F labels at positions 67 (؊0.2 ppm) and 140 (؊0.4 ppm) and downfield changes for positions 248 (؉0.1 ppm) and 316 (؉0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution 19 F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.G-protein-coupled receptors ͉ signal transduction ͉ conformational change ͉ site-directed 19 F labeling ͉ membrane proteins L ight-catalyzed isomerization of 11-cis-retinal in rhodopsin results in a conformational change in the cytoplasmic face. Precise description of this change in rhodopsin and the corresponding conformational changes in G-protein coupled receptors in general on ligand binding is a long range goal of studies on signal transduction. Considerable insights into the tertiary structure in the cytoplasmic face of rhodopsin in the dark and its change on light activation have been obtained recently (1-4). However, none of the approaches used to date can be expected to provide detailed resolution of the tertiary structures involved. Ideally, three-dimensional structures of rhodopsin in the two states are needed. However, neither rhodopsin nor any other G-protein coupled receptor has so far yielded crystals satisfactory for such analysis. Two-dimensional studies using electron diffraction techniques have proven more successful, and these account for the progress made to date in the study of rhodopsin (5). However, the level of resolution remains extremely low for the cytoplasmic and the intradiscal domains.In recent years, NMR spectroscopy has emerged as an alternative powerful approach for studies of protein structure and dynamics (6). While the technique has proved much more fruitful in studies of water-soluble proteins, increasing applications are now being made to hydrophobic polypeptides and membrane proteins (examples in refs. 7-11). However, the latter, because of their size and the necessity of studying them in ...

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Using directed in vitro protein evolution, we generated proteins that bound and antagonized the function of vascular endothelial growth factor receptor 2 (VEGFR2). Binders to human VEGFR2 (KDR) with 10-200 nM affinities were selected by using mRNA display from a library (10(13) variants) based on the tenth human fibronectin type III domain (10Fn3) scaffold. Subsequently, a single KDR binding clone (K(d) = 11 nM) was subjected to affinity maturation. This yielded improved KDR binding molecules with affinities ranging from 0.06 to 2 nM. Molecules with dual binding specificities (human/mouse) were also isolated by using both KDR and Flk-1 (mouse VEGFR2) as targets in selection. Proteins encoded by the selected clones bound VEGFR2-expressing cells and inhibited their VEGF-dependent proliferation. Our results demonstrate the potential of these inhibitors in the development of anti-angiogenesis therapeutics.

The tenth human fibronectin type three domain ((10)Fn3) is a small (10 kDa), extremely stable and soluble protein with an immunoglobulin-like fold, but without cysteine residues. Selections from (10)Fn3-based libraries of proteins with randomized loops have yielded high-affinity, target-specific antibody mimics. However, little is known about the biophysical properties of such antibody mimics, which will determine their suitability for in vitro and medical applications. We characterized target binding and biophysical properties of two related (10)Fn3-based antibody mimics that bind vascular endothelial growth factor receptor two (VEGF-R2). The first antibody mimic, which has a dissociation constant (K(d)) of 13 nM, is highly stable [melting temperature (T(m))=62 degrees C] and soluble, whereas the second, which binds VEGF-R2 with 40 x higher affinity, is less stable (T(m) < 40 degrees C) and relatively insoluble. We used our understanding of these two (10)Fn3 derivatives and of wild-type (10)Fn3 structure to engineer the next generation of antibody mimics, which have an improved combination of high affinity (K(d)=0.59 nM), stability (T(m)=53 degrees C) and solubility. Our findings illustrate that (10)Fn3-based antibody mimics can be engineered for favorable biophysical properties even when 20% of the wild-type (10)Fn3 sequence is mutated in order to satisfy target-binding requirements.

Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.

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G protein-coupled receptors are cell-surface seven-helical membrane proteins that undergo conformational changes on activation. The mammalian photoreceptor, rhodopsin, is the beststudied member of this superfamily. Here, we provide the first evidence that activation in rhodopsin may involve differential dynamic properties of side-chain versus backbone atoms. Highresolution NMR studies of ␣-15 N-labeled receptor revealed large backbone motions in the inactive dark state. In contrast, indole side-chain 15 N groups of tryptophans showed well resolved, equally intense NMR signals, suggesting restriction to a single specific conformation.