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CYANYLATED CYSTEINE AS VIBRATIONAL PROBE OF BINDING-INDUCED STRUCTURAL CHANGES IN

Award Number: R15GM088749
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/30/2009
PERIOD OF PERFORMANCE END DATE: 03/31/2014

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CYANYLATED CYSTEINE AS VIBRATIONAL PROBE OF BINDING-INDUCED STRUCTURAL CHANGES IN - DESCRIPTION (provided by applicant): New experiments are needed to view and to understand the structure of the most dynamic and disordered proteins. Vibrational spectra of proteins can provide this specific information with very fast intrinsic time resolution, which can lead to a snapshot of the entire structural distribution rather than a time-weighted average. Several strategies are proposed here to further develop the C=N stretching vibration of the cyanylated side chain of cysteine as a site-specific probe which can be implemented in dynamic and disordered proteins of arbitrary size. The C=N stretching vibration absorbs in a transparent and uncrowded region of the aqueous biomolecular infrared spectrum, and it can be introduced via post-translational modification at free cysteine side chains. Two systems in which dynamic proteins form structurally uncharacterized bound structures are targeted for study using single site cyanylated cysteine labels. The first is the paramyxoviruses Hendra and Nipah, whose intrinsically disordered NTAIL proteins bind to their P proteins in uncharacterized geometries. The second natural system is calmodulin, a ubiquitous calcium regulatory protein which binds to over 600 different target sequences, with an unusual flexibility in binding and often unknown geometries. Single cyanylated-cysteine-containing variants of each of these proteins will be generated via a mutagenesis/modification scheme in order to map both residue-specific structure formation and contact formation between the protein of interest and possible binding partners. Thermodynamics of binding in the variant proteins will be independently evaluated using isothermal titration calorimetry. New experimental approaches will also be used to place the general application of cyanylated cysteine on a more sure technical footing. These experiments include observation of the C=N stretching band by Raman spectroscopy, placement of C=N inside a semi-collapsed, disordered-yet-bound structure to examine the probe's dependence on water exclusion, and placement of C=N inside the hydrophobic interior of a globular protein. The proposed work will solve two particularly difficult characterization problems in important bound structures, and at the same time, set the stage for wider application of vibrational spectroscopy cyanylated cysteine in many other possible protein systems. PUBLIC HEALTH RELEVANCE: Defects and dysfunction in proteins without well-defined structures, or with dynamic structures and many possible physiological roles, are often associated with systemic human diseases. A novel experimental protocol will be used in this work to reveal atom-level information on binding events in disordered and dynamic proteins, using standard molecular biology techniques and analytical laboratory equipment. The generality of this approach will make it possible to understand, in greater detail, the molecular basis of diseases associated with disordered and dynamic proteins.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2012 ( Subtotal = $303,153 )
2012	2012	HAVERFORD COLLEGE	370 Lancaster Ave	HAVERFORD	PA	19041	MONTGOMERY	USA	Biomedical Research and Research Training	000	2	3/30/2012	COMPETING CONTINUATION	$303,153
														Subtotal = $303,153

Issue Date FY: 2009 ( Subtotal = $202,335 )
2009	2009	HAVERFORD COLLEGE	370 Lancaster Ave	HAVERFORD	PA	19041	MONTGOMERY	USA	Trans-NIH Recovery Act Research Support	000	1	9/22/2009	NEW	$202,335
														Subtotal = $202,335

Grand Total All Awards = $505,488

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CYANYLATED CYSTEINE AS VIBRATIONAL PROBE OF BINDING-INDUCED STRUCTURAL CHANGES IN

Award Number: R15GM088749

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/30/2009

PERIOD OF PERFORMANCE END DATE: 03/31/2014

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