Research projects in our laboratory focus on the investigation of the structure, dynamics, and stability of proteins in unusual environments, for example, in organic solvents. Equipment available in the laboratory to address the scientific issues include modern spectroscopic (e.g., FT-IR, FT-Raman, CD, fluorescence) and analytical (e.g., HPLC, GC) instruments. Examples for some of our research interests are given below.
Structure-guided protein encapsulation. We have recently developed protocols for the encapsulation of proteins in biocompatible polymers minimizing perturbations in their structure. The protein loaded polymer microparticles with dimensions of 10-100 µm in diameter are usually obtained by so-called oil-in-oil, oil-in-water, or water-in-oil-in-water encapsulation procedures. By investigating the protein structure during all critical encapsulation steps to identify detrimental processes (protein inactivation and aggregation), strategies are being developed to systematically eradicate them. As the result, unaltered proteins were delivered from these devices, prerequisite for making them useful in the cure and prevention of human and animal diseases.
Non-aqueous enzymology. Our
laboratory employs enzymes in organic solvents
to catalyze synthetic chemistry reactions. One
problem encountered in such applications is the
frequently very low activity of the enzymes under
such conditions. Consequently, one major goal of
our research is to rationally improve enzyme performance
in organic solvents. Recently, we were successful
in improving the activity and enantioselectivity
of model enzymes by employing methyl-b-cyclodextrin
as activating agent. Mechanistic studies are now
on their way to explain these findings. We are
also involved in research directed to employ enzymes
in the synthesis of drugs or drug precursors. Furthermore,
in collaboration with the group of Dr. Schweitzer-Stenner
the relationship between heme distortion and activity
in peroxidases is being investigated. This investigation
employs poly(ethylene glycol) modified horseradish
peroxidase dissolved in organic solvents.
Interfacial protein denaturation. Hydrophobic
interfaces are known to structurally alter proteins,
frequently leading to irreversible adsorption and
aggregation. This constitutes a major handicap
in many medical applications of protein pharmaceuticals.
Thus, we are working on determining the mechanisms
leading to protein destabilization by hydrophobic
surfaces and interfaces and devise methods allowing protein stabilization
towards this stress. Research
interests in this area also include the use of
enzymes as phase-transfer catalysts.
Protein folding. Recently
our research group became interested in protein
folding phenomena, in particular in the formation
of molten globule intermediates. We have found
that a molten globule state of lysozyme is formed
in the thermal denaturation in glycerol. Currently,
this intermediate is explored with a variety of
spectroscopic techniques. Furthermore, we
embark on a systematic investigation of the structure
and thermal denaturation of other proteins dissolved
in glycerol by CD spectroscopy to characterize
changes in their folding caused by the changes
in the physicochemical environment.
Recent projects. Determination of residual protein structure in the so-called
unfolded state. Enzyme activation by salts. Molecular imprinting. Effect of protein glycosylation on
protein stability and folding.
Selected Publications:
Qing Huang, Qingguo Huang, Roger A. Pinto, Kai
Griebenow, Reinhard Schweitzer-Stenner
and Walter J. Weber, Jr., Inactivation of Horseradish Peroxidase by Phenoxyl Radical
Attack, J. Am. Chem. Soc., 127, 1431-1437 (2005).
Reinhard Schweitzer-Stenner, Thomas Measey, Andrew Hagarman,
Fatma Eker and Kai Griebenow, Salmon Calcitonin and Amyloid β: Two Peptides with Amyloidogenic
Capacity Adopt Different Conformational Manifolds in Their Unfolded States, Biochemistry,
45, 2810-2819 (2006).
Betzaida Castillo, Jessica Mendez, Wasfi Al-Azzam, Gabriel
Barletta and Kai Griebenow, On the relationship between the activity and structure of PEG-α-chymotrypsin conjugates
in organic solvents, Biotechnol. Bioeng., 94, 565-574 (2006).
Betzaida Castillo, Yamaris Pacheco, Wasfi Al-Azzam, Kai
Griebenow, Manjula
Devi, Amaris Ferrer and Gabriel Barletta, On the activity loss of hydrolases in organic
solvents, J. Mol. Catal. B: Enzym., 35, 147-153 (2005).
Andrew Hagarman, Thomas Measey, Ravi S. Doddasomayajula, Isabelle Dragomir,
Fatma Eker, Kai Griebenow and Reinhard Schweitzer-Stenner, Conformational
Analysis of XA and AX Dipeptides in Water by Electronic Circular Dichroism and 1H NMR Spectroscopy, J.
Phys. Chem. B, 110, 6979-6986 (2006).
Ingrid J. Castellanos, Giselle Flores and Kai
Griebenow, Effect of cyclodextrins on α-chymotrypsin stability
and loading in PLGA microspheres upon S/O/W encapsulation, J. Pharm. Sci., 95, 849-858 (2006).
Ricardo J. Sola and Kai
Griebenow, Chemical glycosylation:
New insights on the interrelation between protein structural mobility, thermodynamic stability,
and catalysis, FEBS Lett., 580, 1685-1690 (2006).
Ingrid J. Castellanos,
Giselle Flores and Kai
Griebenow, Effect
of the molecular weight of poly(ethylene glycol)
used as emulsifier on α-chymotrypsin stability upon encapsulation
in PLGA microspheres, J. Pharm. Pharmacol.,
57, 1261-1269 (2005).
Wasfi Al-Azzam, Emil A. Pastrana, Brian King, Jessica Mendez and
Kai Griebenow, Effect of the covalent modification of horseradish peroxidase with
poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres, J. Pharm. Sci., 94, 1808-1819 (2005).
Thomas Measey, Andrew Hagarman, Fatma Eker, Kai
Griebenow and Reinhard
Schweitzer-Stenner, Side Chain Dependence of Intensity and Wavenumber Position of Amide
I' in IR and Visible Raman Spectra of XA and AX Dipeptides, J. Phys. Chem. B, 109,
8195-8205 (2005).
Ingrid J. Castellanos, Wasfi Al-Azzam and Kai
Griebenow, Effect
of the covalent modification with poly(ethylene glycol) on α-chymotrypsin stability
upon encapsulation in poly(lactic-co-glycolic) microspheres, J. Pharm. Sci., 94, 327-340 (2005).
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