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Structure and Dynamics in Solutions and Membranes 

Overview
Macromolecular crystallography has been enormously successful in elucidating the structures of proteins and other biomolecules. These data are being combined with emerging genetic and biochemical information on pathways to suggest temporal, spatial, and functional relations controlling cellular function.

However, the central question in biophysics still remains: What is the connection between the structure and function of biological macromolecules such as proteins, DNA, RNA, polysaccharides, and their complexes? Answering this question requires understanding the dynamics of macromolecular structures in their natural environment, where flexibility of the molecules and water, pH, and ion concentration play determinant roles. Examples include protein and nucleic acid folding and unfolding, polymer collapse upon change of solvent, electron transfer, and large-scale fluctuations in macromolecules.

Studies of the structure and dynamics of molecules in solutions and membranes are an essential complement to macromolecular crystallography. Such studies are poised to provide new insights into the function, control, and dynamics of individual macromolecules and large molecular complexes. Solutions and membranes are the media where many of the most intriguing and complex biological processes take place, including molecular recognition, signal transduction, chemical sensing, transport, synthesis, degradation, replication, and defense.

The high brightness of NSLS-II will give x-ray scattering and spectroscopy studies of biomolecules and membranes unparalleled sensitivity and time resolution, enabling more precise structure determinations and extending measurements of dynamics down to the microsecond time range.

Protein Folding
Understanding protein folding, i.e. how a protein achieves its stable functional three-dimensional structure from a linear string of amino acids, is key to understanding how the protein performs its biological functions. Time resolved solution scattering now allows researchers to follow the structural changes of the protein as folding proceeds.

The time scale of the folding process varies from picoseconds to nanoseconds (when the initial secondary structure starts to form) to milliseconds to seconds (when the folding process is completed). With the development of more accurate force fields that simulate the interactions that govern the folding process, and construction of more powerful computers, computational biologists are lengthening their calculations to the time scale of microseconds. The high brightness of NSLS-II will extend the time resolution of solution-scattering measurements down to this time scale. Computations and experiments will then overlap and provide better tests of our understanding of the physics of the underlying interactions.

Structural Kinetics in Biological Macromolecular Complexes
High-resolution analysis methods, such as x-ray crystallography and nuclear magnetic resonance (NMR), have difficulty dealing with macromolecular complexes, where structural kinetics, such as changes in shape, may accompany the biological function that is under study.

Low-resolution structural modeling of the bacteriophage PRD1 vertex complex restored from solution scattering data, viewed from different angles. both the structuresof the individual components and the overall structure of the complex are obtained from analysis of solution scattering data.

In contrast, small angle solution scattering can provide structural information that reveals global conformation changes in biomolecules and molecular complexes in solution, and is thus complementary to protein crystallography. Solution scattering is an effective technique for determining the low-resolution, basic shape of these complexes (see figure). This approach also holds great potential for resolving the structural kinetics of macromolecular complexes using time resolved solution scattering to produce low-resolution movies of events such as the assembly and operation of molecular machines.

The high brightness of NSLS-II will provide high intensity, highly collimated, and small x-ray beams, resulting in high quality data necessary for accurate shape determinations. NSLS-II will also enable time resolved measurements at microsecond time resolutions.

Extending Solution Scattering to Membrane Proteins
In addition to proteins, non-crystalline cellular membrane structures can also be studied with x-ray scattering. Individual membrane protein molecules diffuse within the confinement of the lipid bilayer - the greasy component of the membrane, which is much like a two dimensional (2D) liquid that can be investigated with small-angle x-ray scattering (SAXS) (see figure).

Scattering from a two-dimensional liquid of membrane pores (model shown in the middle). Neutron scattering can easily detect these structures in multiple-layered model membranes (left), whereas only much more ordered liquids were observed by x-rays (right). The increased intensity of NSLS-II will make up for the contrast disadvantage of x-ray scattering.

All of the solution scattering techniques used for proteins are equally applicable to the 2D liquid of membrane proteins, including low-resolution shape determination and time resolved measurement. The high brightness of NSLS-II will enable solution scattering experiments to provide information about these structural transitions. These phenomena are related directly to the function of the membrane proteins, and therefore are central to many branches of life-science research.

Views of the structure of the T. therm ophilius 70S ribosome from the back of the 30S subunit. Structures like the 70S ribosome have relied both on synchrotron studies and cryo-electron microscopy results.

Counterion Cloud
In their native environment biomolecules are surrounded by water and ions. While it is known that the availability of water and ions is an important factor in protein structure and function, little is known about the structure of "ordered" water and the distribution of counterions around biomolecules. Counterions neutralize the biomolecules, preventing charge-charge interactions between them. Using synchrotron radiation it is possible to specifically probe the counterion cloud while keeping the biomolecules in solution. The high brightness of NSLS-II in the 1 to 4 keV energy range makes it very well suited to use anomalous scattering techniques on biologically relevant ions such as Na (1.07 keV), Mg (1.3 keV), Cl (2.8 keV), K (3.6 keV), Ca (4.0 keV), S (2.5 keV) and P (2.1 keV).

Study of Metalloproteins with X-ray Absorption Spectroscopy
X-ray absorption spectroscopy (XAS) can be used to measure the transition of core electronic states of a metal atom to excited electronic states. Spectral analysis near this transition, x-ray absorption near-edge structure (XANES), provides information on the metal's charge state and geometry. Spectral analysis above the absorption edge (up to 10-15% above the edge in energy), extended X-ray absorption fine structure (EXAFS) provides complementary structural information, such as numbers, types, and distances of ligands or neighboring atoms. Both techniques are valuable for studying a variety of metal sites in biological systems.

Traditionally, XAS, like most other spectroscopies, has been used as a static probe of structure. NSLS-II provides the opportunity to extend x-ray absorption measurements into the time-resolved realm, potentially revolutionizing the study of biochemical and bioinorganic systems. For example, proteins containing metal, called metalloproteins (hemoglobin is an example), react over extremely quick time scales - sometimes as fast as a picosecond. NSLS II will allow much faster time scales to be probed than XAS is currently capable of.

Electron Delocalization in Biomolecules
The wave nature of the electron determines almost all of the properties of simple condensed matter, such as color, mechanical hardness, electrical resistivity, thermal conductivity, thermal expansion, dielectric constant, and melting and boiling points. Thus, it is worth investigating how electron delocalization underlies protein function, since proteins are, after all, a form of condensed matter. NSLS-II will enable a new class of such phenomena to be studied for the first time.

Left: a schematic representation of the photosynthetic apparatus in the intra-cytoplasmic membrane of purple bacteria. Right: a model for the pigment-protein complexes in the modeled bacteria rhodobacter sphaeroides.

As an example, consider the light harvesting complex (LHC) protein-chromophore (see figure) of the photosynthetic bacterium rhodobacter sphaeroides. Light is first absorbed by a circular aggregate of bacteriochlorophyll molecules and carotenoids held together by proteins, LH-II. Energy is then transferred to a circular antenna complex, LH-I, surrounding the reaction center, and finally to the reaction center (RC) where it starts the respiratory cycle of the cell. The structures of LH-II and RC are known to atomic resolution from x-ray diffraction experiments. However, the mechanism and dynamics of electron energy transfer remains a mystery.

The high brightness and flux of NSLS-II, together with new optical schemes, will enable inelastic x-ray experiments that will open new windows into the electronic properties of biomolecules.

 

Last Modified: May 2, 2014
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