| One of the cornerstones of structural biology is the link between the protein sequence and its structure and function.Several techniques can monitor protein structure and dynamics with different degrees of spatial and time resolution. X-ray crystallography can determine their static structure with atomic resolution, but requires the availability of crystals, whereas many interesting systems such as the protein aggregates mentioned above are non-crystalline (also, X-rays cannot monitor dynamics). NMR also provides 3D structural information with atomic resolution, but it is a rather complex and time consuming procedure involving several phases, such as data collection, resonance assignment,restraint generation and final structure calculation and refinement, making it unsuitable for rapidly screening different point mutations or solution conditions for a particular protein. A full structure determination typically takes many days of experimental time and weeks of analysis. A variety of optical techniques can be used to study the secondary structure of proteins and to distinguish between different motifs. These include UV and IR absorption, UV and IR circular dichroism (CD) and UV resonance Raman scattering. Such approaches are much less time-consuming, but provide only partial spectroscopic information and cannot address more subtle differences in secondary structure.A very recent series of theoretical studies has highlighted the potential of 2DUV spectroscopy to provide information on the secondary structure of proteins, exciton dynamic, protein orientation and secondary structure composition. 2DUV can be considered as an extension of UV absorption and CD spectroscopies, in which the spectra are spread along a second frequency axis, greatly enhancing the information content and structural sensitivity. An advantage of 2DUV is that it avoids isotope labelling, by monitoring the electronic nπ*/ππ*transitions of the backbone (and their coupling) or using the three relatively rare aromatic residues (tryptophan, tyrosine,phenylalanine) as local probes.It is one of the aims of this project to establish 2DUV spectroscopy as a diagnostic tool for structural and dynamic studies of polypeptides and proteins. We expect that this method will have the directness and speed of operation of optical techniques but with a much greater information content, bridging the experimental gap between crude estimates of protein unfolding and full structure determination, and thus enabling rapid assessment of which protein variants or sample preparations are worthwhile torgets of more involved structural and dynamic studies.Electronic excitations of these chromophores depend on their surroundings through electrostatic interactions, making them good local probes for structures.The nπ* and ππ* transitions of the peptide backbone are in the far UV (190-250 nm)and are often used as global probes of the protein secondary structure. The amino acids with aromatic side chains, on the other hand, display distinct absorption bands in the near UV (250-300 nm) range. There are only three out of twenty such amino acids: tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). Since aromatic amino acids are relatively rare in proteins (approximately 4% Phe, 3% Tyr, 1.5% Trp occurrence probability) they can be used as built-in local structural probes without the need for isotope labelling.Understanding the exciton dynamics in biological systems is crucial for the manipulation of their function. We present a combined quantum mechanics (QM) and molecular dynamics (MD) simulation study that demonstrates how coherent two dimensional near-ultraviolet (2DNUV) spectra can be used to probe the exciton dynamics in a mini-protein, Trp-cage. The 2DNUV signals originate from aromatic transitions that are significantly affected by the couplings between residues, which determine exciton transport and energy relaxation. The temporal evolution of 2DNUV features captures important protein structural information, including geometric details and peptide orientations. We have used the temporal evolution of 2DNUV spectra to study the structural-dependent exciton dynamics in a model protein. We demonstrated that the exciton transport and energy relaxation rate depend on the structural parameters of the protein, such as the geometric details and peptide orientations. These would be very useful for the structural determination of proteins and reveal some crucial structure-property relationships. One can also expect some anisotropy of motions and allosteric behavior in proteins, which will help understand and manipulate biochemically relevant interactions such as ligand binding so that facilitate related drug designs.Quantitative measurements of protein orientation and secondary structure composition are of great importance for protein biotechnology applications and disease treatments, and yet, they are technically challenging for a spectroscopic study.On the basis of quantum mechanics/molecular mechanics simulations, we demonstrate that two dimensional (2D) linear dichroism spectroscopy is capable of probing the direction of secondary structure motifs in proteins. In addition, by calculating the ratio of transverse ππ* signals to longitudinal ππ* signals in 2D spectra, we can achieve quantitative measurement of the fraction of a-helix content in a protein. The quantitative determination of secondary structures in proteins is one of the important protein structure analysis. We showed that 2DUV zzzz and 2D LD signals can monitor the orientation changes of helical, sheet, and amyloid fibril protein structures. Different orientation dependences for different structural motifs have been established simultaneously. Compared to one-dimensional spectra, 2D spectra provide us more information associated with the orientation of structures, suggesting a path for higher resolution spectroscopy. We also got the content fraction of helical structures from the analysis of 2DUV zzzz signals as a function of the orientation. This information is helpful to describe a fine picture of protein structure. Our work may open up new windows for probing amyloid fibril structures and dynamical fluctuations and associated photochemical and photophysical processes, which are crucial to understand and manipulate their functionality. |
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