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        Molecular Bioprocessing Research
 
 

 

 

 

  

The Journal of Physical Chemistry B

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"Molecular Simulations of Multimodal Ligand-Protein Binding: Elucidation of Binding Sites and Correlation with Experiments"

Freed, AS, Garde S, Cramer SM

2011, 115 (45), pp 13320-13327

B&B cover image

Biotechnology and Bioengineering

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"Investigation of Protein Binding Affinity and Preferred Orientations in Ion Exchange Systems Using a Homologous Protein Library"

Chung WK, Hou Y, Freed A, Holstein M, Makhatadze GI, Cramer SM

2009, 102 (3), pp 869-881

 
 

        About Dr. Cramer

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Professor Steven Cramer is the William Weightman Walker Professor of Polymer Engineering at Rensselaer Polytechnic Institute, Troy, New York. He is currently conducting research on several areas related to protein-surface interactions and molecular bioprocessing. In addition, Professor Cramer is known worldwide for his expertise in separations in general. He is the Editor-in-Chief of the International journal Separation Science and Technology. Professor Cramer was awarded the Alan S. Michaels Award for the Recovery of Biological Products (ACS Division of Biochemical Technology). He was also awarded Rensselaer's School of Engineering Research Excellence Award, a Presidential Young Investigator award from the National Science Foundation, the Early Career Award from Rensselaer Polytechnic Institute as well as several teaching awards. Professor Cramer has been elected a fellow of the American Insitute of Chemical Engineers, the American Insitute for Medical and Biological Engineering and the American Chemical Society. He has also chaired several prestigious meetings including several International HIC/RPC Bioseparations Conferences, the ACS Recovery of Biological Products Meeting and the Gordon Conference on Reactive Polymers. Professor Cramer has published over 150 papers in peer-reviewed journals and has 9 patents. Importantly, he has produced 36 Ph.D. students who have gone on to leadership positions in the biotechnology industry and academia.

      Our Research

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The research in the Cramer laboratory involves state of the art experimental and theoretical investigations into novel bioseparations systems. Topics include: prediction of protein binding affinity and multiscale modeling of chromatographic systems, design of chemically selective displacers, development of efficient antibody separation systems, fundamental studies in multimodal chromatography, novel lab on chip separations systems, protein unfolding in chromatographic systems, chemometrics for process analytical technology, multilevel automated peptide synthesis/screening system for design of affinity peptides and biosensors, smart biopolymer affinity precipitation systems, use of high throughput screening for quality by design of large scale bioprocesses, design of novel affinity reagents for purification of therapeutic enzymes, and development of hierarchical nano-bio materials for bioprocessing. As one example, recent work that combines protein libraries, NMR studies, molecular dynamics simulations and high throughput chromatographic investigations has provided significant insight into the design of a new generation of mixed mode chromatographic systems.

Recent Highlights:

Journal of Physical Chemistry B
Molecular Simulations of Multimodal Ligand-Protein Binding: Elucidation of Binding Sites and Correlation with Experiments 
Freed, AS, Garde, S, Cramer, SM.

2011, 115 (45), pp 13320-13327
Abstract:
Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

Biotechnology and Bioengineering
Investigation of Protein Binding Affinity and Preferred Orientations in Ion Exchange Systems Using a Homologous Protein Library
Chung, WK, Hou, Y, Freed, A, Holstein, M, Makhatadze, GI, Cramer, SM
2009, 102 (3), pp 869-881
A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure-property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various Mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems

 

 

   

Summer 2012    

 

 

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