IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Mapping solvent accessibility in proteins.
Autor/es:
GÓMEZ, G.E; MONTI, J.L; DELFINO J.M
Reunión:
Conferencia; International Gregorio Weber Conference.; 2011
Resumen:
Much knowledge into protein folding, ligand binding and
complex formation can be derived from the examination of the nature and size of
the accessible surface area (ASA) of the polypeptide chain, a key parameter in
protein science not directly measurable in an experimental fashion. To this end,
an ideal chemical approach should aim at exerting solvent mimicry to probe the
protein surface regardless of its chemical nature. The choice of the
photoreagent diazirine (DZN) to fulfill these goals arises from its size
comparable to water and from being an ideal source of the extremely reactive
methylene carbene (:CH2). This species inserts readily into any X-H bond, yielding
stable methylated products. The tritiated form of DZN (3H-DZN) has been
employed to address unfolding transitions of single-domain proteins1-3 and the
contact surface of an antigen-antibody complex4. By taking advantage of the
superb sensitivity and high resolution of modern mass spectrometry techniques (MS)
we introduce an alternative (non-radioactive) quantitative signal proportional
to the extent of modification (EM) of the sample. The urea-induced
conformational transition experienced by α-lactalbumin was monitored by
following the EM signal by ES MS, alongside known conformational probes such as
circular dichroism and intrinsic fluorescence emission5. To address the issue
of identifying labeled sites along the polypeptide chain, enzymatic
fragmentation at the level of small peptides was achieved, thus enabling the
construction of a map of solvent accessibility. Moreover, by subsequent MALDI
MS/MS analysis of peptides we demonstrate the feasibility of attaining amino
acid resolution at defining the target sites reached by the DZN reagent. Patterns
of methylation obey Poisson statistics, validating an interpretation of local
labeling whereby the observed modification at a given fragment results from the
sum of independent probabilities of reaction at each site. Overall, DZN
labeling coupled to MS analysis offers a unique perspective on protein
conformation, shedding light on native and non-native states as well as on
protein complexes.