The National Institute of Diabetes & Digestive & Kidney Diseases

We are investigating the relationships between protein structure, dynamics, and function using ultrafast time-resolved laser spectroscopy and X-ray crystallography. These experimental techniques employ an ultrashort laser "pump" pulse (shorter than 10^-13 seconds) to trigger a photophysical or photochemical reaction and a variably delayed "probe" pulse that measures the spectral or structural evolution of the protein. This pump-probe technique provides the means to acquire "snapshots" of a protein as it executes its designed function. By monitoring the changes that occur over time, from femtoseconds to milliseconds, we aim to build a foundation for understanding how proteins execute their designed tasks with high efficiency and selectivity.

To probe the structural evolution of an entire protein as it executes its designed task, we have developed the technique of picosecond time-resolved X-ray crystallography. This effort is part of a multinational collaboration with experiments carried out on Michael Wulff’s time-resolved beam line at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France. In 2003, we reported the first 150 picosecond time-resolved X-ray structure of a myoglobin mutant in Science. Since then, we have improved the experimental methodology and have produced detailed 3D movies of proteins as they function. Model systems being investigated include ligand-binding heme proteins and their mutants, bacteriorhodopsin, and photoactive yellow protein.

Time-resolved spectroscopic methods spanning wavelengths from the uv to the IR are used to probe the dynamics of specific functional groups with ultrafast time resolution. A microfocusing femtosecond spectrometer has been developed that is capable of probing protein dynamics in small crystals. By comparing dynamics in solution and crystals, we can assess the extent to which crystal contacts influence protein dynamics. Moreover, ultrafast studies of protein photophysics have led to more efficient protocols for chromophore photoactivation, thereby improving the quality of time-resolved X-ray structures.

Our crystallographic and spectroscopic studies are performed on a large number of molecules and reveal ensemble-averaged changes. To generate a molecular level understanding of how proteins function, we would prefer to watch a protein function one molecule at a time. While it is impossible to experimentally follow structural changes in a single molecule with ultrafast time resolution, that feat can be achieved computationally. In a collaboration with Dr. Gerhard Hummer at the NIH, we have performed a joint analysis of time-resolved X-ray structures and ensemble-averaged molecular dynamics (MD) simulations. Validated by the stunning agreement – in both space and time – of the simulation average and detailed time-resolved X-ray structures, the single-molecule trajectories give us access not only to the transient intermediates of the experiments, but also to the transitions between them. From this single-molecule perspective, we were able to show at atomic resolution how conformational switches control ligand migration through a transiently interconnected network of internal cavities, and how protein dynamics can modulate functional ligand binding. We continue to employ a combination of spectroscopic, crystallographic, and computational methods to generate new and deeper insights into mechanisms of protein function.

1. Anderson S, Srajer V, Pahl R, Rajagopal S, Schotte F, Anfinrud P, Wulff M, Moffat K Chromophore conformation and the evolution of tertiary structural changes in photoactive yellow protein. Structure (Camb) (12): 1039-45, 2004. [Full Text/Abstract]

2. Lim M, Jackson TA, Anfinrud PA Orientational distribution of CO before and after photolysis of MbCO and HbCO: a determination using time-resolved polarized Mid-IR spectroscopy. J Am Chem Soc (126): 7946-57, 2004. [Full Text/Abstract]

3. Schotte F, Soman J, Olson JS, Wulff M, Anfinrud PA Picosecond time-resolved X-ray crystallography: probing protein function in real time. J Struct Biol (147): 235-46, 2004. [Full Text/Abstract]

4. Hummer G, Schotte F, Anfinrud PA Unveiling functional protein motions with picosecond x-ray crystallography and molecular dynamics simulations. Proc Natl Acad Sci U S A (101): 15330-4, 2004. [Full Text/Abstract]

5. Plech A, Wulff M, Bratos S, Mirloup F, Vuilleumier R, Schotte F, Anfinrud PA Visualizing chemical reactions in solution by picosecond x-ray diffraction. Phys Rev Lett (92): 125505, 2004. [Full Text/Abstract]

6. Bourgeois D, Vallone B, Schotte F, Arcovito A, Miele AE, Sciara G, Wulff M, Anfinrud P, Brunori M Complex landscape of protein structural dynamics unveiled by nanosecond Laue crystallography. Proc Natl Acad Sci U S A (100): 8704-9, 2003. [Full Text/Abstract]

7. Schotte F, Lim M, Jackson TA, Smirnov AV, Soman J, Olson JS, Phillips GN Jr, Wulff M, Anfinrud PA Watching a protein as it functions with 150-ps time-resolved x-ray crystallography. Science (300): 1944-7, 2003. [Full Text/Abstract]

8. Sagnella DE, Straub JE, Jackson TA, Lim M, Anfinrud PA Vibrational population relaxation of carbon monoxide in the heme pocket of photolyzed carbonmonoxy myoglobin: comparison of time-resolved mid-IR absorbance experiments and molecular dynamics simulations. Proc Natl Acad Sci U S A (96): 14324-9, 1999. [Full Text/Abstract]

9. Gai F, Hasson KC, McDonald JC, Anfinrud PA Chemical dynamics in proteins: the photoisomerization of retinal in bacteriorhodopsin. Science (279): 1886-91, 1998. [Full Text/Abstract]

10. Lim M, Jackson TA, Anfinrud PA Ultrafast rotation and trapping of carbon monoxide dissociated from myoglobin. Nat Struct Biol (4): 209-14, 1997. [Full Text/Abstract]

11. Hasson KC, Gai F, Anfinrud PA The photoisomerization of retinal in bacteriorhodospin: experimental evidence for a three-state model. Proc Natl Acad Sci U S A (93): 15124-9, 1996. [Full Text/Abstract]

12. Lim M, Jackson TA, Anfinrud PA Binding of CO to myoglobin from a heme pocket docking site to form nearly linear Fe-C-O. Science (269): 962-6, 1995. [Full Text/Abstract]

13. Lim M, Jackson TA, Anfinrud PA Nonexponential protein relaxation: dynamics of conformational change in myoglobin. Proc Natl Acad Sci U S A (90): 5801-4, 1993. [Full Text/Abstract]