Since macromolecules are considered to be dynamic, structural alterations such as side chain motions or movement of large domains are believed to play an important role in their function and stability. Our center is equipped with a series of instruments that can measure aspects of the dynamic behavior of proteins under various environmental variables conditions of pH, ionic strength, temperature, agitation, and other variables.
- HR-US 102 ultrasonic spectrometer
- Microcal VP-DSC with pressure perturbation capability
- Fluorescence Innovations MATRIX-UV lifetime spectrometers
- Red-edge excitation fluorescence measurements (Olis Protein Machine or PTI fluorometers)
- Agilent TOF mass spectrometer for H/D exchange studies (Dr. David Weis lab, Dept. of Chemistry at KU)
Proteins in the presence of deuterium will undergo an exchange of amide hydrogen atoms for deuterium atoms at a rate dependent on the dynamic exposure of the peptide backbone. This rate of exchange is a function of both solvent penetration and local unfolding. For this reason, extended measurements of the rate of hydrogen/deuterium exchange can be related to the small amplitude motions within the protein that render interior regions accessible to the solvent. Mass spectrometry or FTIR can be used to measure the rate of exchange of the different classes of protein amide protons. The mass spectrometry studies are performed in collaboration with Dr. Weis of the Chemistry department at KU.
High Resolution Ultrasonic Spectroscopy
Ultrasonic spectroscopy functions by exposing protein samples to an oscillating pressure wave and measuring the change in its velocity and attenuation. The difference or relative change in ultrasonic velocity and/or attenuation between a reference and sample cell is plotted as a function of temperature or time. Protein molecules often contain many cavities, which cause internal motions or flexibility responses to thermal and mechanical forces. These cavities can be compressed, providing an important measure of flexibility, volume fluctuations, and stability. Sharp increases in the attenuation accompany protein aggregation, and decreases in the wave velocity correspond to unfolding or increased compressibility of the protein.
Pressure Perturbation Calorimetry
In this method, a dilute solution of protein and a blank sample are subjected to pulses of pressure as heat is transferred to or from the samples to maintain a constant temperature. The differential heat change between the sample and the reference cell can be used to calculate the coefficient of thermal expansion (α) based on thermodynamic principles. Similarly, the relative expansibility, hydration, and volume changes of unfolding can be monitored.
Time-Correlated Single Photon Counting (TCSPC)
TCSPC is a spectroscopic technique that measures the fluorescence intensity and anisotropy decays associated with fluorophores of interest (such as trp or dyes). TCSPC is a digital technique that counts photons, which are time-correlated in relation to an excitation pulse. The sample is repetitively excited using a femtosecond pulse from a Ti:sapphire laser, and a histogram of photon arrival times is used to represent the intensity decay of the sample. The shape of the anisotropy decay contains information about the geometry, reorientational motions, and flexibility of the molecule. A variety of dynamic molecular information within the range of picosecond to nanosecond timescales can be obtained using this technique.
Red Edge Excitation Shift Spectroscopy (REES)
Red edge excitation is a characteristic property of polar fluorophores that exhibit excitation wavelength-dependent emission spectra. This phenomenon depends upon motional restriction of the environment of fluorophores (intrinsic and extrinsic). The red edge excitation shifts are affected by changes in solvent relaxation around an excited state fluorophore, which in turn is influenced by dynamic motions within proteins and solvent fluctuations around the environment of the fluorophore. The magnitude of red edge effect can therefore be a sensitive probe to monitor dynamics and flexibility in the local environment around a fluorophore of interest.
Temperature-Dependent UV-Absorbance Peak Shifts
Second derivative ultraviolet spectroscopy is a sensitive technique to study alterations in protein dynamics by precise measurements of ultraviolet absorbance peak shifts in aromatic residues of proteins in the pre-transition regions. These peak shifts can be induced both by changes in temperature and by the presence of salt cations such as Li+, Na+ or Cs+. The magnitude and direction of the peak shifts can be affected by different factors such as protein size and charge, dynamics of the local environment, and solvent accessibility of the aromatic residues. This technique can complement other dynamic measurements and provide additional information about protein dynamics in the pre-transition regions before any detectable unfolding event occurs.
Ramsey, J.D., Gill, M.L., Kamerzell, T.J, Price, E.S., Joshi, S.B., Bishop, S.M., Oliver, C.N. and Middaugh, C.R. (2009) Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability. J Pharm Sci. 2009 Jul; 98(7): 2432-47
PMID 19072858 http://www.ncbi.nlm.nih.gov/pubmed/19072858