The problem of protein folding and dynamics: Time-resolved dynamic nonradiative excitation energy transfer measurements

Elisha Haas

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    20 Scopus citations


    The proteins are molecular "machines" that are self-assembled infinitely diverse, give rise to complex structures of any size, and have well-controlled vectorial modes of conformational changes, driven by thermal motions. Numerous experiments show that the pathway of folding of globular proteins, from the unordered state to the native conformations, and their modes of intramolecular motions, are determined by the sequence of the amino acyl residues (the monomers) in the polypeptide chain (the genetic information) of each protein. Attempts to decipher the mechanism by which the multiple transitions, on a wide range of time and distance scales are directed, depend on development of new methods for determination of distributions of intermolecular distances in flexible molecules and their time dependence. Methods based on time resolved measurements of intramolecular dynamic nonradiative excitation energy transfer, combined with protein engineering and site directed labeling were developed. Global analysis of data sets obtained by these measurements yield distributions of probabilities of segments end-to-end distances (EEDP) (in the range of 8 to 80 Å chain). Methods for determination of rates of conformational fluctuations and of the folding transitions, down to picosecond time scale, by means of this approach were also developed. Application of this experimental approach in the study of the denatured state of single-domain globular proteins, show that even in the denatured states, these proteins have compact structures. The bovine pancreatic trypsin inhibitor (BPTI) showed in the denatured state at least two conformational subpopulations, one was native-like and the other was characterized by EEDP distributions corresponding to an unfolded polypeptide chain. Specific nonlocal interactions of a pair or a cluster of residues, are thought to bring together ends of specific segments, and thus accelerate the folding transition. A new device for measurements of the time dependence of changes of the EEDP distributions of globular proteins during the folding transition (the "double kinetics" method) was developed. Multidomain enzyme molecules were shown to undergo specifically directed conformational transitions coupled to substrate binding. The EEDP distributions measured for the enzyme adenylate kinase, a tri-domain enzyme that catalyzes the phosphorylation of AMP molecules, show that in solution the apo-enzyme does not have a single conformation, but rather undergoes slow exchange between multiple structures, including that of the closed conformation. Analysis of the transitions coupled to substrates and inhibitor binding show sequential reduction of the means of the EEDP distributions, and increase of the rigidity of the protein structures. These measurements show that the domain closure transition of the phototransferase enzymes are combinations of many conformational changes, and not rigid body motions. Both intra- and inter-domain structural dynamics is essential for this type of transition. Current developments of both protein engineering and tunable pulsed laser light sources are expected to further enhance the effectiveness of these applications, with improved resolution and finer mesh of distance determination.

    Original languageEnglish
    Pages (from-to)1088-1106
    Number of pages19
    JournalIEEE Journal of Selected Topics in Quantum Electronics
    Issue number4
    StatePublished - Dec 1996

    Bibliographical note

    Funding Information:
    Manuscript received December 2, 1996; revised January 10, 1997. This work was supported under Grants from the National Institutes of Health of General Medical Sciences, the United States–Israel Binational Science Foundation (BSF), under Equipment Grants and Research Grants from the Fund for Basic Research of the Israeli Academy of Sciences, the Ministry of Science of the Government of Israel, and the Russell Foundation of Miami Beach, FL.


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