Susan G. Frasier

Bio: Susan G. Frasier is an academic researcher. The author has contributed to research in topics: Computer program & Non-linear least squares. The author has an hindex of 1, co-authored 1 publications receiving 429 citations.

[16] Nonlinear least-squares analysis

Abstract: Publisher Summary This chapter highlights one of the methods for the analysis of experimental data, along with the assumptions, advantages, and disadvantages of the method. The most important experimental detail to understand for any parameter estimation procedure is the sources and magnitudes of the random and nonrandom experimental errors superimposed on the data. Moreover, computers are not oracles. The investigator needs to be continually aware that the output of any computer program is no better than what goes into it. It is important to realize that a computer is in essence the same as any other instrument in a laboratory. To obtain optimal results it must be used correctly. To use it correctly, the user must understand the assumptions which went into the development of the computer programs and their limitations.

How to measure and predict the molar absorption coefficient of a protein.

Abstract: The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:3193261 and is based on data from Edelhoch [1967, Biochemistry 6:1948-19541.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, 6 M guanidine hydrochloride (GdnHCI), and 8 M urea have been measured. For Trp, the average E values for the proteins are less than the E values measured in any of the solvents. For Tyr, the average E values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured t values for 80 proteins, the t at 280 nm of a folded protein in water, t(280), can best be predicted with this equation:

Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding.

Abstract: Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (delta Cp), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and delta Cp.

Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants.

Abstract: Characteristics and properties of the unfolding free energy change, delta G degrees N-U, as determined by the linear extrapolation method are assessed for the unfolding of phenylmethanesulfonyl chymotrypsin (PMS-Ct). Difference spectral measurements at 293 nm were used to define PMS-Ct unfolding brought about with guanidinium chloride, urea, and 1,3-dimethylurea. All three denaturants were shown to give identical extinction coefficient differences (delta epsilon N-U) between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of delta epsilon N-U on denaturant supports the linear extension of pre- and postdenaturational base lines into the transition zone, allowing evaluation of unfolding equilibrium constants based on the two-state assumption. An expression, based on the linear extrapolation method, was used to provide estimates of delta G degrees N-U for the three denaturants using nonlinear least-squares fitting of the primary data, delta epsilon versus [denaturant]. The three delta G degrees N-U values were identical, within error, suggesting that the free energy change is a property of the protein system and independent of denaturant. It is suggested that the error in delta G degrees N-U determined from use of the linear extrapolation method is significantly larger than commonly reported in the literature.

Circadian rhythms in cultured mammalian retina.

Abstract: Many retinal functions are circadian, but in most instances the location of the clock that drives the rhythm is not known. Cultured neural retinas of the golden hamster (Mesocricetus auratus) exhibited circadian rhythms of melatonin synthesis for at least 5 days at 27 degrees celsius. The rhythms were entrained by light cycles applied in vitro and were free-running in constant darkness. Retinas from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 hours, showed a shortened free-running period of melatonin synthesis. The mammalian retina contains a genetically programmed circadian oscillator that regulates its synthesis of melatonin.

Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth hormone secretion in man.

Abstract: Studies in man have shown that the episodic release of growth hormone (GH) is infrequent and erratic, and unlike that in the rat does not appear to have discernible ultradian periodicities. However, these observations in nonfasted subjects may be invalid since mixed nutrients have unpredictable effects on GH release. Moreover, in the fed state basal GH levels are frequently undetectable, thus rendering the identification of low amplitude pulses unreliable. Accordingly, the 24-h pulsatile pattern of GH secretion obtained from repetitive venous sampling in six normal adult male subjects was examined during a control fed day and during the first and fifth days of a 5-d fast. The GH data were analyzed using two distinct methods: a discrete pulse detection algorithm (Cluster analysis) and Fourier expansion time-series, which allows fixed periodicities of secretory activity to be resolved. The 5-d fast resulted in a significant increase in discrete GH pulse frequency (5.8 +/- 0.7 vs. 9.9 +/- 0.7 pulses/24 h, P = 0.028), 24 h integrated GH concentration (2.82 +/- 0.50 vs. 8.75 +/- 0.82 micrograms.min/ml; P = 0.0002), and maximal pulse amplitude (5.9 +/- 1.1 vs. 12.3 +/- 1.6 ng/ml, P less than 0.005). While multiple low-amplitude sinusoidal periodicities were present on the control fed day, time-series analysis revealed enhancement of circadian and ultradian cycles on the first and fifth days of fasting. Concomitantly, fasting resulted in a decline (day 1 vs. day 5) in serum concentrations of somatomedin C (1.31 +/- 0.22 vs. 0.77 +/- 0.18 U/ml) and glucose (4.9 +/- 0.2 vs. 3.2 +/- 0.2 mmol/liter), and a marked rise in free fatty acid (0.43 +/- 0.12 vs. 1.55 +/- 0.35 mmol/liter) and acetoacetate (35 +/- 6 vs. 507 +/- 80 nmol/liter). We conclude that the acute nutritional status is an important determinant of spontaneous pulsatile GH secretion in man. Fast-induced enhancement of GH release is achieved through combined frequency (discrete pulses) and amplitude (sinusoidal periodicities) modulation. Such alterations in somatotropic hormone release may play an important role in substrate homeostasis during starvation.