Luminescence analysis of the structure of human alpha-1-microglobulin

Absorption, fluorescence, and fluorescence excitation spectra in UV and visible regions are studied for alpha-1-microglobulin preparations isolated from human urine by gel chromatography and immunoaffinity chromatography with charcoal adsorption. The possible nature of low-molecular-weight compounds that impart yellow-brown color to alpha-1-microglobulin preparations and their role in the stabilization of the structure of protein globule is discussed. The effect of urea (1–10 M) and guanidine hydrochloride (0.25–6 M) on the conformational state and fast internal dynamics of alpha-1-microglobulin is studied by tryptophan fluorescence. The unfolding of the protein under the action of denaturants is attended with pronounced activation of its nanosecond internal dynamics. Alpha-1-microglobulin can regain the initial conformation and internal dynamics typical of native protein after denaturation unfolding of the globule with 10 M urea or 6 M guanidine hydrochloride. Alpha-1-microglobulin isolated by gel chromatography can exist in a partially folded thermodynamically stable state in 4–6 M urea.     

Effect of temperature on the internal dynamics and the conformational state of bacterial alkaline phosphatase

Room-temperature tryptophan phosphorescence and fluorescence have been used to study the slow internal dynamics and the conformational state of Escherichia coli alkaline phosphatase in the temperature range from 0 to 100°C. The heating of alkaline phosphatase solution within the 0–70°C range has been shown to amplify considerably the internal dynamics. The further raise of temperature to 95°C brings about a reversible increase in the internal dynamics and partial unfolding of the globule. The heating of protein solution within a narrow temperature range of 97–100°C gives rise to irreversible conformational transition with complete globule unfolding, sharp amplification of the internal dynamics, and loss of enzymatic activity.     

Effect of temperature on intramolecular dynamics and conformational state of bacterial alkaline phosphatase

The slow internal dynamics and the conformational state of Escherichia coli alkaline phosphatase by the action of temperature in the range 0-100 degrees C have been investigated by tryptophan room temperature phosphorescence and fluorescence. It has been shown that heating an alkaline phosphatase solution in the interval 0-70 degrees C leads to a substantial increase in the slow internal dynamics. A further increase in temperature to 95 degrees C causes a reversible enhancement of internal dynamics and a partial unfolding of the globule. Heating the protein solution in a narrow temperature range 97-100 degrees C induces an irreversible conformational transition, which is characterized by total unfolding of the globule, a drastic increase in internal dynamics, and the loss of enzymatic activity.     

On the possibility for a protein to exist in a manifold of partially folded states

With the help of the methods of tryptophan fluorescence and room-temperature phosphorescence and using Escherichia coli alkaline phosphatase as an example, the ability of a protein to exist in a manifold of partially folded thermodynamically stable states differing in conformation, the internal dynamics, and functional activity was shown. Such intermediate (between native and unfolded) structures may form during unfolding or folding of the protein. It was shown that the degree of destruction of the native structural organization of the globule depends on both the nature and the mode of action of the destroying agent and the structure of the protein. Conformational transitions of the globule can change the kind of the internal dynamics (fast, slow), and shifts of dynamics can initiate conformation changes of the protein and precede them. A scheme of the structural and functional transformations of the protein during denaturation is presented, which takes into account the possibility of globule transitions into a manifold of functional active and inactive partially folded states. The role of partially folded forms of cell proteins in the development of pathology is discussed.     

Luminescent analysis of the structure of human alpha-1-microglobulin

The spectra of absorption, fluorescence, and excitation of fluorescence of preparations of alpha-1-microglobulin isolated from human urea by two methods, gel chromatography and immunoaffinity chromatography with additional purification by activated charcoal, have been investigated in ultraviolet and visible regions. A possible nature of low-molecular compounds coloring alpha-1-microglobulin yellow-brown and their role in stabilizing the structure of protein globule are discussed. The action of urea (1.0-10 M) and guanidine hydrochloride (0.25-6 M) on the conformational state and the fast (nanosecond) internal dynamics of alpha-1-microglobulin has been investigated by the method of tryptophan fluorescence. It has been shown that the unfolding of alpha-1-microglobulin under the action of these denaturants is associated with a significant increase in the nanosecond internal dynamics of protein. The ability of alpha-1-microglobulin to restore the initial conformation characteristic for the native protein and the internal dynamics after the unfolding of the globule by 10 M urea and 6 M guanidine hydrochloride has been ascertained. It has been found that alpha-1-microglobulin isolated by the method of gel chromatography can exist in solution of 4-6 M urea in a thermodynamically stabile partialy folded state.