In situ atomic force microscopy (AFM) is an exceedingly powerful and useful technique for characterizing the structure and assembly of proteins in real-time, in situ, and especially at model membrane interfaces, such as supported planar lipid bilayers. There remains, however, a fundamental challenge with AFM-based imaging. Conclusions are inferred based on morphological or topographical features. It is conventionally very difficult to use AFM to confirm specific molecular conformation, especially in the case of protein-membrane interactions. In this case, a protein may undergo subtle conformational changes upon insertion in the membrane that may be critical to its function. AFM lacks the ability to directly measure such conformational changes and can, arguably, only resolve features that are topographically distinct. To address these issues, we have developed a platform that integrates in situ AFM with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This combination of tools provides a unique means of tracking, simultaneously, conformational changes, not resolvable by in situ AFM, with topographical details that are not readily identified by conventional spectroscopy. Preliminary studies of thermal transitions in supported lipid bilayers and direct evidence of lipid-induced conformational changes in adsorbed proteins illustrates the potential of this coupled in situ functional imaging strategy.
A number of recent experiments and simulations strongly support the idea that dynamics are spatially heterogeneous in glassy materials. Relaxation times appear to be correlated over a few nanometers, supportive of the notion of cooperatively-rearranging-regions (CRR) containing of order 100 molecules. But details of the local cooperative dynamics are still mysterious. Certain issues, such as the heterogeneity lifetime, and whether local relaxation within a CRR is exponential, remain controversial. I will describe experiments in which molecular cooperativity was directly observed near the glass transition, through nanoscale probing of dipolar noise in polymer glasses. The dynamics and evolution of individual CRR was studied. Surprisingly, individual CRR were found to revisit a handful (2-4) of configurations up to hundreds of times. Statistical analysis of the noise gives information about the lifetime of the CRR, the local shape and evolution of the energy landscape, and the evolution from exponential to nonexponential response within a CRR.
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