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Binding site for the C‐domain of insulin‐like growth factor (IGF) binding protein‐6 on IGF‐II; implications for inhibition of IGF actions

“…NMR data were processed in XWINNMR version 3.1 (Bruker Biospin) and analyzed using XEASY version 1.3 [18]. A pH of 3.5 was used for the direct observation of 15 N– 1 H HSQC spectra of 15 N‐labelled IGF‐II as these spectra show significant line broadening at higher pH [19] and this is the lowest pH at which binding still occurs. In preliminary experiments, 15 N– 1 H HSQC spectra of 0.3 mM 15 N‐labelled C‐BP‐6 in the presence of 0.3 mM unlabelled IGF‐II were recorded at pH 4.5, 4.0, 3.5 and 3.0.…”

“…Therefore, pH 3.5 was selected for experiments investigating the C‐BP‐6 binding site on IGF‐II. IGF‐II 15 N– 1 H assignments were based on those of Torres et al [19]. The majority of cross‐peaks from IGF‐II backbone amides showed some line broadening upon addition of unlabelled C‐BP‐6.…”

Site‐specific examination of secondary structure and orientation determination in membrane proteins: The peptidic 13C18O group as a novel infrared probe

“…3 More recently, site directed dichroism employing 13 CA 16 O isotope edited peaks has been used to obtain structures for a variety of transmembrane ␣-helical bundles such as influenza A virus M2, 8 HIV vpu, 19 , influenza C virus CM2, 20 and human phospholamban. 7 The method relies on the fact that one is able to accurately measure the dichroism in two or more isotope edited peaks (each contained within a different sample). With this information in hand, it is then possible to numerically solve the coupled equations relating the measured dichroism to the helix tilt, rotational pitch angle and fractional sample order, ␤, , and f, respectively.…”

“…We have previously described the application of the above probe. 7 Herein we describe in detail all aspects regarding its synthesis, analysis, and possible utilizations.…”

Secondary structure determination of 15N‐labelled human Long‐[Arg‐3]‐insulin‐like growth factor 1 by multidimensional NMR spectroscopy

“…This Gly‐30‐Ala‐39 region of native IGF‐I was poorly defined in the homonuclear studies and did not display any solvent protection [11, 13], thus it is not clear whether this is a real difference between Long‐[Arg‐3]‐IGF‐I and IGF‐I or a limitation of the homonuclear studies. IGF‐II has a smaller stretch of residues in an extended conformation between helix 1 and helix 2 that are also protected from solvent exchange but, compared to Long‐[Arg‐3]‐IGF‐I, this region is offset five residues toward the N‐terminus at Gly‐25 to Phe‐28 [22]. A long stretch of residues in the N‐terminal extension (Met‐13 to Leu‐4) is also protected from solvent.…”