Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

Abstract

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...

Isofagomine Induced Stabilization of Glucocerebrosidase

“…[7] Here we probe the effects of IFG-binding in solution on GCase global stability by differential scanning fluorimetry [18][19][20][21][22] and on local dynamics by amide hydrogen/ deuterium exchange coupled with proteolysis and mass spectrometry (H/D-Ex). [23][24][25][26][27][28] The ability to partially restore intracellular trafficking and lysosomal GCase localization of mutant forms of GCase was then inferred from activity assays performed on N370S/N370S and F213I/L444P patient fibro-blasts. These experiments were employed to gain a better understanding of the PC mechanism associated with IFG binding, particularly the relationship between local dynamics, global stability in solution and their effect on lysosomal concentrations of the enzyme.…”

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

“…The cooperative binding of oxygen to Hb is a particu-larly important area of research. Although the basic principles underlying the oxy (R) to deoxy (T) transition have been elucidated several decades ago [20], new details regarding the mechanism of these structural switching events continue to emerge [21][22][23][24].…”

The Role of Protein Dynamics in Increasing Binding Affinity for an Engineered Protein−Protein Interaction Established by H/D Exchange Mass Spectrometry

“…In this regard, the hydrogen/deuterium exchange technique (H/D-Ex) is particularly powerful through its ability to identify transient fluctuations in protein molecules or protein-protein interfaces (11)(12)(13). This is because the rate of exchange of a backbone amide hydrogen in the interior of a protein molecule is a direct function of the energy required to distort the polypeptide chain to provide access to the deuterated solvent.…”

“…Neutron diffraction studies have also established that high-resolution H/D exchange patterns can be determined from a time-averaged analysis (12), but the technique is fraught with experimental barriers (14). More recently, mass spectrometry methods have been developed and employed to accurately determine H/D exchange patterns for proteins (15)(16)(17)(18)(19)(20), protein-small molecule complexes (13,21,22), and protein-protein complexes (23)(24)(25). While this method does not have the single-residue resolution of NMR, it is has the advantage of speed and the ability to analyze larger proteins and protein complexes.…”