The structure of ␣-synuclein (␣-syn) amyloid was studied by hydrogen-deuterium exchange by using a fragment separation-MS analysis. The conditions used made it possible to distinguish the exchange of unprotected and protected amide hydrogens and to define the order͞disorder boundaries at close to amino acid resolution. The soluble ␣-syn monomer exchanges its amide hydrogens with water hydrogens at random coil rates, consistent with its natively unstructured condition. In assembled amyloid, long N-terminal and C-terminal segments remain unprotected (residues 1-Ϸ38 and 102-140), although the N-terminal segment shows some heterogeneity. A continuous middle segment (residues Ϸ39 -101) is strongly protected by systematically H-bonded cross-␤ structure. This segment is much too long to fit the amyloid ribbon width, but non-H-bonded amides expected for directionchanging loops are not apparent. These results and other known constraints specify that ␣-syn amyloid adopts a chain fold like that suggested before for amyloid-␤ [Petkova et al. M any proteins and polypeptides are able to adopt the generic massively aggregated structure known as amyloid (1). The macroscopic fibrillar character of amyloid is obvious by direct electron microscopic observation, but its detailed structure and the structural basis of its unusual behavior remains a challenging problem (2-9).Methods based on the hydrogen exchange (HX) behavior of polypeptides can provide useful information. The backbone amide hydrogens of proteins engage in continual exchange with the hydrogens of solvent water. These hydrogens, uniformly distributed at every amino acid (except proline) in every protein molecule, provide built-in, structure-sensitive, nonperturbing probes that can be used to study soluble or insoluble proteins under any desired conditions. Hydrogens that are freely exposed to solvent exchange at known rates that depend on pH, temperature, neighboring residues, and the hydrogen isotopes used (10,11). Hydrogens that are protected by structure, almost always in H bonds, exchange far more slowly. Their exchange is modulated by dynamic structural events that reversibly separate protecting H bonds and transiently expose them to the normal chemical exchange process. Accordingly, HX measurements can distinguish the presence and absence of protecting structure, determine the thermodynamic stability and dynamic properties of local and surrounding structure, and probe the effects of mutations, manipulations, and conditions, in principle, at amino acid resolution (12).The development of multidimensional solution NMR methods (13-15) has made high-resolution analysis of HX behavior routine for small soluble proteins (16). A fragment separation method that does not depend on solution NMR measurement extends HX studies to large proteins and insoluble protein systems (17)(18)(19). In this method, hydrogen isotope exchange can be performed under conditions that are most pertinent for the protein system being studied. Timed samples are then placed into slow HX condition...

An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.H ydrogen exchange (HX) measurements can, in principle, locate protein-binding sites and structure changes and can quantify otherwise unavailable dynamic and energetic parameters (1-4). For relatively small proteins, HX can be measured at an amino acid resolved level by NMR methods. For larger, functionally more interesting proteins, other strategies are required. Earlier work (5, 6) developed a ''functional-labeling'' approach that can selectively label, by hydrogen-tritium (H-T) or hydrogen-deuterium (H-D) exchange, just those sites that change in any functional process. In favorable cases, the label can then be located at medium resolution by a proteolytic fragmentation method in which the fragments are quickly produced and then separated by HPLC under conditions where the loss of isotopic label is slow (6-9).To move toward higher resolution and more comprehensive coverage of target proteins, recent work in many laboratories has coupled the HPLC separation to a second dimension of fragment resolution by online MS (10, 11). These methods tend to be labor intensive and time consuming, with limitations in throughput and comprehensiveness and in the structural resolution of functionally important changes. This article merges previous HX functional labeling and fragment separation methods with an automated MS approach termed deuterium exchange MS (DXMS) (12-18).We are using Hb as a model system to study how protein molecules manage intramolecular signal transduction processes. Hb functions by transducing a part of the binding energy of its initially bound O 2 ligands into structure-change energy. The energy is carried through the protein to distant heme sites in the form of energetic structure changes, and there transduced back into binding energy. The initial reduced binding energy and the later enhanced binding produces the physiologically important sigmoid binding curve. In short, the currency of allosteric interactions is free energy. Trying to understand allostery without measuring free energy is like trying to understand an economic system without measuring money. A great deal of information on regulatory structure change in many proteins is now available, but mainly in a qualitative pictorial sense from ''before and after'' crystallographic or NMR views. How these changes participate in energy transduction and translocation has been little explored (19)(20)(21...