ABSTRACT
UV absorption spectroscopy is commonly used with peptides for determining concentration and enzyme activity, but high-resolution UV spectra can also provide information on peptide secondary and tertiary structure and association behavior. New developments using temperature- and cation-dependent high-resolution second derivative absorption methods can also provide information concerning peptide dynamics. Data from several low-resolution spectroscopic techniques, including UV absorption, can be combined to generate an overall picture of peptide structure as a function of environmental conditions.
Subject(s)
Peptides/chemistry , Spectrophotometry, Ultraviolet/methods , Absorption , Data Interpretation, Statistical , Kinetics , TemperatureABSTRACT
In peptides, steady-state fluorescence can be used to measure the intrinsic fluorescence of Trp, Tyr, and Phe residues, as well as associated dyes, which can change upon exposure to different environmental conditions. This technique can be thus used to detect changes in the conformational states of peptides. Time-resolved fluorescence can also be used to study fast motions of peptides and their interactions with fluorescence dyes. Data from several low-resolution spectroscopic techniques, including fluorescence, can be combined to generate an overall picture of peptide structure as a function of environmental conditions.
Subject(s)
Peptides/chemistry , Spectrometry, Fluorescence/methods , Angiotensin I/chemistry , Statistics as Topic , Substance P/chemistry , Time Factors , alpha-MSH/chemistryABSTRACT
Circular dichroism measures the difference between the absorbance of left- and right-handed circularly polarized light, and can be used to monitor the secondary structure of peptides (far UV) and the tertiary structure of larger polypeptides (near UV). This technique is especially useful for helix-coil transitions and other aspects of structural alterations. Data from several low-resolution spectroscopic techniques, including CD, can be combined to generate an overall picture of peptide structure as a function of environmental conditions.
Subject(s)
Circular Dichroism/methods , Peptides/chemistry , Angiotensin I/chemistry , Data Interpretation, Statistical , Substance P/chemistry , alpha-MSH/chemistryABSTRACT
Fourier transform infrared (FTIR) spectroscopy provides data that are widely used for secondary structure characterization of peptides. A wide array of available sampling methods permits structural analysis of peptides in diverse environments such as aqueous solution (including optically turbid media), powders, detergent micelles, and lipid bilayers. In some cases, side chain vibrations can also be resolved and used for tertiary structure and chemical analysis. Data from several low-resolution spectroscopic techniques, including FTIR, can be combined to generate an empirical phase diagram, an overall picture of peptide structure as a function of environmental conditions that can aid in the global interpretation of large amounts of spectroscopic data.
Subject(s)
Peptides/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Data Interpretation, Statistical , Islet Amyloid Polypeptide/chemistry , Protein Structure, SecondaryABSTRACT
Internalization of G-protein-coupled receptors is mediated by phosphorylation of the C-terminus, followed by binding with the cytosolic protein arrestin. To explore structural factors that may play a role in internalization of cannabinoid receptor 1 (CB1), we utilize a phosphorylated peptide derived from the distal C-terminus of CB1 (CB1(5P)(454-473)). Complexes formed between the peptide and human arrestin-2 (wt-arr2(1-418)) were compared to those formed with a truncated arrestin-2 mutant (tr-arr2(1-382)) using isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The pentaphosphopeptide CB1(5P)(454-473) adopts a helix-loop conformation, whether binding to full-length arrestin-2 or its truncated mutant. This structure is similar to that of a heptaphosphopeptide, mimicking the distal segment of the rhodopsin C-tail (Rh(7P)(330-348)), binding to visual arrestin, suggesting that this adopted structure bears functional significance. Isothermal titration calorimetry (ITC) experiments show that the CB1(5P)(454-473) peptide binds to tr-arr2(1-382) with higher affinity than to the full-length wt-arr2(1-418). As the observed structure of the bound peptides is similar in either case, we attribute the increased affinity to a more exposed binding site on the N-domain of the truncated arrestin construct. The transferred NOE data from the bound phosphopeptides are used to predict a model describing the interaction with arrestin, using the data driven HADDOCK docking program. The truncation of arrestin-2 provides scope for positively charged residues in the polar core of the protein to interact with phosphates present in the loop of the CB1(5P)(454-473) peptide.
Subject(s)
Arrestins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Receptor, Cannabinoid, CB1/chemistry , Receptor, Cannabinoid, CB1/metabolism , Amino-Acid N-Acetyltransferase , Arrestins/genetics , Arrestins/isolation & purification , Humans , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Conformation , Sequence Deletion , beta-Arrestin 2 , beta-ArrestinsABSTRACT
Desensitization of the cannabinoid CB1 receptor is mediated by the interaction with arrestin. In this study, we report the structural changes of a synthetic diphosphorylated peptide corresponding to residues 419-439 of the CB1 C-terminus upon binding to arrestin-2. This segment is pivotal to the desensitization of CB1. Using high-resolution proton NMR, we observe two helical segments in the bound peptide that are separated by the presence a glycine residue. The binding we observe is with a diphoshorylated peptide, whereas a previous study reported binding of a highly phosphorylated rhodopsin fragment to visual arrestin. The arrestin bound conformations of the peptides are compared.
