Dynamics of parvalbumin studied by fluorescence emission and triplet absorption spectroscopy of tryptophan.
Fluorescence emission and triplet-triplet absorbance spectroscopy of the single tryptophan in cod parvalbumin were used to study the stability and dynamics of the protein as influenced by Ca2+ binding and interaction with a chaotropic agent. The concentrations for half-saturation for Ca binding were 3.6 x 10(-9), 3.3 x 10(-4), 7.1 x 10(-3), and 0.14 M in the presence of 0, 2, 3, and 4 M guanidine hydrochloride, respectively. As predicted for thermodynamic reversibility, the guanidine hydrochloride unfolding reaction depends upon Ca2+, and the delta G are as follows: 22.9, 29.3, 35.2, and 44.2 kJ/mol for no added Ca2+, 1, 2, and 5 mM Ca2+, respectively. The stability toward denaturation imparted by the binding of two Ca2+ is about -60 kJ/mol. For Ca(2+)-bound parvalbumin in the presence of excess Ca2+, the decay of the triplet state tryptophan is approximately exponential, and the lifetime decreases from 6.5 to 3.8 ms as the temperature increases from 10 to 40 degrees C. In contrast, the triplet decay of the calcium-free protein is nonexponential over the time range of microseconds to milliseconds, a result that may indicate that the Ca-free protein is molten-globule-like. At Ca2+ concentrations where the protein is partially saturated with Ca2+, the lifetime of the longest decay component is less than that for the Ca-saturated protein; this finding suggests an exchange of Ca2+ and a conformational change during the triplet lifetime. From these data, a rate constant for the process that includes calcium-related protein conformational change can be surmised to range between 200 and 500 s-1.
Sudhakar
K
K
Department of Biochemistry & Biophysics, School of Medicine of Pennsylvania, Philadelphia 19104.
Phillips
C M
CM
Owen
C S
CS
Vanderkooi
J M
JM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D005399:Fishes; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D007700:Kinetics; D008163:Luminescent Measurements; D010100:Oxygen / Q000737:chemistry; D010320:Parvalbumins / Q000737:chemistry*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D006146:Guanidines; D010320:Parvalbumins; D014364:Tryptophan; D019791:Guanidine; D010100:Oxygen; D002118:Calcium