Only very recently has the availability of intense synchrotron sources enabled smaller and less perfect crystals, hence growable under less exigent conditions, to be measured, and has also permitted snapshots and rapid sequences of snapshots to be taken, providing time-resolved structural information. But this work is still quite preliminary.
Alternative approaches have, therefore, been tirelessly sought, in which proteins could be observed in their native environments and sequences of structural changes resolved temporally.
One of the most promising of the new approaches has been single-molecule observation techniques developed in a number of laboratories, mainly in Japan, in which a label (e. g. a fluorophore) is attached to the protein, and high resolution optical microscopy used to observe its motions. This impressive work has led to very significant new insights into the working mechanism of molecular motors being obtained (Yanagida et al. 1993, Funatsu et al. 1995, Ishijima et al. 1998). Note that many of the problems investigated in this field are connected with proteins at some kind of interface, most typically the solid/liquid interface, where the solid part may be a bilayer liquid membrane, and the liquid an aqueous fluid mimicking the cytoplasm.
Much interest has also been shown in the atomic force microscope (AFM), but it seems fair to say that its most successful application has been for the imaging of regular arrays, to which averaging and refinement can be applied to achieve truly molecular resolution (as it is of course with XRD). Although there are examples showing how AFM of individual proteins can provide important corroborative information, there is not the same strong line of inference from the raw measured data to the desired molecularly resolved information as exists for XRD; analysis often boils down to the subjective assessment of images, and high resolution kinetics is problematic.
With both these techniques (single-molecule observation and AFM), although the protein is measured in a native environment, the molecule is severely perturbed. Although the label required in the fluorescent technique maybe small relative to the entire protein molecule (and even this is often not the case), it is likely to be large relative to some functionally important part. With AFM, the contact between the cantilever-mounted tip and the protein is akin to using an elephant to move a football—irreparable damage to the latter is likely to result.
Hence the search for non-invasive, native-compatible and dynamically sensitive techniques continues. Note that both the single-molecule observation and AFM techniques require that the protein under investigation is present at a solid/liquid interface. Since the majority of proteins in their native state operate at such an interface, most commonly the surface of a bilayer lipid membrane, this is not a restricting condition.
The purpose of this chapter is to show that another new technique, well adapted to the study of proteins at or near interfaces, namely high resolution optical waveguide lightmode spectroscopy (OWLS), is capable of providing structural information superior to that obtainable from AFM, with excellent time resolution, in native environments, and without the need for labels. It is well known that even though the "ruler" of optical methods is of the order of a wavelength, i.e. hundreds of nanometres for visible radiation, interferometry can resolve structural changes with subnanometre resolution and nanosecond time resolution (Rembe et al. 2005). In the same way, OWLS, also known as high resolution molecular microscopy (HRMM), can also achieve subnanometre resolution of key average characteristic lengths of molecular systems.
The basic experimental arrangement is to place the interfacial region under examination on a planar optical waveguide. For example, if the interaction of a protein with lipid bilayers is being studied, the waveguide is coated with the bilayer, which is placed in contact with a dilute solution of the protein.
The structure of the remainder of this chapter is as follows: The second section focuses on OWLS, describing the fundamentals of reflectance-based methods for interrogating objects at an interface in general and OWLS in particular (briefly contrasting it with other optical methods); the next section outlines the practical determination of the lightmode spectrum; the section on static structures examines how static structural parameters of adlayers are obtained; the following section discusses kinetic analysis and dynamic structural inference; and the last section discusses advanced topic in kinetic analysis and dynamic structural inference.
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