Analysis Method and Experimental Conditions Affect Computed Circadian Phase from Melatonin Data

Accurate determination of circadian phase is necessary for research and clinical purposes because of the influence of the master circadian pacemaker on multiple physiologic functions. Melatonin is presently the most accurate marker of the activity of the human circadian pacemaker. Current methods of analyzing the plasma melatonin rhythm can be grouped into three categories: curve-fitting, threshold-based and physiologically-based linear differential equations. To determine which method provides the most accurate assessment of circadian phase, we compared the ability to fit the data and the variability of phase estimates for seventeen different markers of melatonin phase derived from these methodological categories. We used data from three experimental conditions under which circadian rhythms - and therefore calculated melatonin phase - were expected to remain constant or progress uniformly. Melatonin profiles from older subjects and subjects with lower melatonin amplitude were less likely to be fit by all analysis methods. When circadian drift over multiple study days was algebraically removed, there were no significant differences between analysis methods of melatonin onsets (P = 0.57), but there were significant differences between those of melatonin offsets (P<0.0001). For a subset of phase assessment methods, we also examined the effects of data loss on variability of phase estimates by systematically removing data in 2-hour segments. Data loss near onset of melatonin secretion differentially affected phase estimates from the methods, with some methods incorrectly assigning phases too early while other methods assigning phases too late; missing data at other times did not affect analyses of the melatonin profile. We conclude that melatonin data set characteristics, including amplitude and completeness of data collection, differentially affect the results depending on the melatonin analysis method used

Hydrogen Bond Strengths in Phosphorylated and Sulfated Amino Acid Residues

<div><p>Post-translational modification by the addition of an oxoanion functional group, usually a phosphate group and less commonly a sulfate group, leads to diverse structural and functional consequences in protein systems. Building upon previous studies of the phosphoserine residue (pSer), we address the distinct nature of hydrogen bonding interactions in phosphotyrosine (pTyr) and sulfotyrosine (sTyr) residues. We derive partial charges for these modified residues and then study them in the context of molecular dynamics simulation of model tripeptides and sulfated protein complexes, potentials of mean force for interacting residue pairs, and a survey of the interactions of modified residues among experimental protein structures. Overall, our findings show that for pTyr, bidentate interactions with Arg are particularly dominant, as has been previously demonstrated for pSer. sTyr interactions with Arg are significantly weaker, even as compared to the same interactions made by the Glu residue. Our work sheds light on the distinct nature of these modified tyrosine residues, and provides a physical-chemical foundation for future studies with the goal of understanding their roles in systems of biological interest.</p> </div

Characterization of Hydrogen Bonds to Glu, pSer, pTyr, and sTyr in Experimental Protein Structures.

<p>Columns report the percentage of hydrogen bonds to Glu, pSer, pTyr or sTyr, that are to a given donor residue, and for Arg in a single or bidentate orientation. Residues were drawn from all structures in the Protein Databank containing a pSer, pTyr, or sTyr residue. For Glu, residues were taken from the set of structures containing a pSer residue.</p

A comparison of electrostatic potentials for sTyr, pTyr(−1), and pTyr(−2).

<p>Electrostatic potentials are shown at isosurfaces of +/−2 kTe. The protonated phosphate group of pTyr(−1) presents a shaped charge that can provide a stronger interaction with hydrogen bond donors than the more isotropic charge on the sTyr sulfate.</p

Hydrogen Bond Occupancies in Molecular Dynamics Simulation of Tripeptide Systems.

<p>Columns report the percentage of frames showing a particular hydrogen bond for tripeptides Xxx-Gly-Yyy where Xxx represents hydrogen bonding donors Arg, Lys, or Gln; and Yyy represents Glu, pSer(−2), pSer(−1), pTyr(−2), pTyr(−1), or sTyr. For Arg tripeptides, the table reports percentages for single and bidentate interactions.</p

Implicit Solvent Potentials of Mean Force for representative residue pairs.

<p>Plots show interaction energy vs. distance, at distance intervals of 0.25 Å, for a pair of residues in a given orientation. Distance refers to the P-C distance between the phosphate atom and the terminal carbon on Arg (coplanar), or the N-O distance (collinear).</p

Percentage of Glu, pSer, pTyr and sTyr residues showing a given number of hydrogen bonds.

<p>Residues were drawn from all structures in the Protein Databank containing a pSer, pTyr, or sTyr residue. For Glu, residues were taken from the set of structures containing a pSer residue.</p

Change in phase estimates (hours) for subjects with 2-hour gaps of data.

<p>Change in phase estimates for simulations of 2-hour missing data at different times relative to the melatonin phase marker for five subjects total from Studies 1, 2 and 3. Each panel plots the change in phase estimates for a different method. Data gaps are referenced as the time of midpoint of each gap relative to the DLMOpost-threshold computed from the complete data set for each subject. Positive changes in phase estimate indicate that the estimated phase from the missing data set is later relative to the estimate from the complete data set, while negative values indicate that the estimate from the missing data set was earlier.</p

Phase Markers from Six Method Types of Analyzing Melatonin Data.

<p>Diagram of the phase markers from six methods used to analyze a single melatonin profile (subject 1849v). The upper panel includes the assayed melatonin values, plotted as melatonin concentration (pmol/L). The lower panel groups the various phase markers by method and indicates their position with respect to upper panel. Fourier-based analysis methods F(2) and F(3) have been combined in this diagram because of their similarity.</p

Variability of all methods With Drift and Drift Removed.

<p>The Box plots show Maximum, Mean, Minimum, and Percentiles 99, 75, 50, 25, and 1 of the data from all subjects. Black are onset methods, red are offset methods, and blue are maximum or midpoint methods. Note difference in y-axis scales between With Drift and Drift Removed.</p