个人简介

Ph.D., Massachusetts Institute of Technology B.S., Brown University

研究领域

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Structural Biology, Protein Chemistry, Biophysical Signal Processing

Prof. Makowski’s research focus is on the development and use of innovative biophysical methods for the study of complex biomolecular systems. His current research interests include use of x-ray solution scattering and neutron spin-echo spectroscopy for the study of proteins in solution including characterization of protein-protein and protein-ligand interactions and structural fluctuations; the use of coherent diffraction imaging for the study of crystalline fibrils in complex biological tissues, with particular emphasis of cellulose in plant cell walls. Wide-angle x-ray solution scattering: X-ray solution scattering in both the small-angle (SAXS) and wide-angle (WAXS) regimes is making an increasing impact on our understanding of biomolecular complexes. The accurate calculation of WAXS patterns from atomic coordinates has positioned the approach for rapid growth. WAXS data are sensitive to small structural changes in proteins; useful for calculation of the pair-distribution function at relatively high resolution; provides a means to characterize the breadth of the structural ensemble in solution; and can be used to identify proteins with similar folds. WAXS data are often used to test structural models, identify structural similarities and characterize structural changes. WAXS is highly complementary to crystallography and NMR. It holds great potential for the testing of structural models of proteins; identification of proteins that may exhibit novel folds; characterization of unfolded or natively disordered proteins; and detection of structural changes associated with protein function. Current work is aimed at characterizing the structure of intermediates in enzyme catalysis and structural transformations – catching images of proteins while they are at work. Protein Ensembles: It is becoming increasingly clear that characterization of the protein ensemble – the collection of all conformations of which the protein is capable – will be a critical step in developing a full understanding of the linkage between structure, dynamics and function. X-ray solution scattering in the small angle (SAXS) and wide-angle (WAXS) regimes represents an important new window to exploring the behavior of ensembles. The characteristics of the ensemble express themselves in x-ray solution scattering data in predictable ways. We have collected and analyzed WAXS data on a range of biomolecular systems and demonstrated the modulation of these ensembles by ligand binding; mutation and environmental factors. Neutron Spin Echo Spectroscopy: Neutron spin-echo spectroscopy is an underutilized probe of protein dynamics sensitive to slow correlated motions (including translational and rotational diffusion and internal modes) on the picosecond to nanosecond time scale. We have used neutron spin-echo (NSE) spectroscopy to study structural fluctuations that occur in hemoglobin (Hb) and myoglobin (Mb) in solution. Using NSE data to very high momentum transfer, q ( ~ 0.62 Å-1), the internal dynamics of these proteins were characterized at the level of the dynamical pair correlation function and self-correlation function in the time range of several picoseconds to a few nanoseconds. Comparison of data from the two homologous proteins collected at different temperatures and protein concentrations was used to assess the contributions to the data made by translational and rotational diffusion and internal modes of motion. The temperature dependence of the decay times can be attributed to changes in viscosity and temperature of the solvent as predicted by the Stokes-Einstein relationship. This is true for contributions from both diffusive and internal modes of motion indicating an intimate relationship between the internal dynamics of the proteins and the viscosity of the solvent. Viscosity change associated with protein concentration can account for changes in diffusion observed at different concentrations, but is apparently not the only factor involved in the changes in internal dynamics observed with change in protein concentration. Data collected at high q indicate that internal modes in Mb are generally faster than in Hb, perhaps due to the greater surface to volume ratio of Mb and the fact that surface groups tend to exhibit faster motion than buried groups. Comparison of data from Hb and Mb at low q indicate an unexpectedly rapid motion of the hemoglobin αβ-dimers relative to one another. Dynamic motion of subunits is increasingly perceived as important to the allosteric behavior of hemoglobin. Our data demonstrate that this motion is highly sensitive to protein concentration, temperature and solvent viscosity, indicating that great care need be exercised in interpreting its effect on protein function. Nanoscale Architecture of Plant Cell Walls: Lignocellulose in plants represents the largest renewable source of organic molecules on earth. Utilization of this resource is hindered by the nanoscale architecture of the plant that limits accessibility of chemical bonds to external reagents. Working with plant biologists at Purdue and National Renewable Energy Laboratory, we are using a broad range of x-ray scattering and imaging techniques to characterize the structure of plant cell walls on the nano- to meso-scales. X-ray phase contrast imaging; x-ray fluorescence microscopy; small- and wide-angle x-ray scattering; and coherent diffraction imaging are being used to characterize the deconstruction of plant cell walls during chemical and thermal pre-treatment procedures. Dilute acid treatment leads to disorientation and disorganization of large scale structures but does not substantially disrupt the fundamental building blocks – the elementary fibrils of cellulose. Supplemental iron – a potential catalyst for lignocelluloses breakdown – co-locates specifically with cellulosic structures. Continuing research focuses on refining processing methods and enhancing the methods used, including development of scanning x-ray microdiffraction.