GOMEZ Gabriela Elena
congresos y reuniones científicas
Mapping solvent accessibility in proteins.
Conferencia; International Gregorio Weber Conference.; 2011
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.