256 A
A N A LY T I C A L C H E M I S T R Y / M A Y 1 , 2 0 0 1
A powerful new approach that goes goe
John R. Engen ¥ David L. Smith University of Nebras -
T
horough characterization of a protein requires understanding the interplay of three separate but linked features—function, structure, and dynamics. Investigating these features demands a wide range of analytical techniques. For example, characterizing protein function, which is the identification of a protein’s role in cellular maintenance, is usually more amenable to biochemical, molecular, and cellular types of analyses. On the other hand, studies of protein structure and dynamics most often use physical methods, many of which are common to analytical chemistry. © HAMID GHANADAN/GH MULTIMEDIA 2001
M A Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
257 A
(a) H
H
H
H
H
H
CON CH CON CH CON CH CON CH CON CH CON CH2
CH2
CH2
CH2
(CH2)4
CONH2
COOH
OH
SH
NH2
Asn
Asp
Ser
Cys
Lys
Structure and dynamics
(b)
H
D D
H
(c) D2O + OD– k1
k–1 UH
NH k–1
k2
UD
ND k1
OD– + HOD
FIGURE 1. Amide hydrogen exchange in peptides and proteins. (a) Hydrogens used for exchange studies. (b) Protein unfolding dynamics associated with isotope exchange. (c) Model used to link isotope exchange with protein unfolding dynamics.
It was recognized nearly 50 years ago that different protein structures undergo isotope exchange at different rates, and this has led to widespread use of hydrogen exchange as a means to study protein structure and dynamics. Hydrogen exchange has been used to follow protein folding, detect structural changes due to mutation, and study protein–protein interactions. Isotope exchange studies have most often used tritium or deuterium with analysis by nuclear magnetic resonance (NMR), IR, or UV spectroscopies. Over the past decade, hydrogen/deuterium (H/D) exchange
258 A
A N A LY T I C A L C H E M I S T R Y / M A Y 1 , 2 0 0 1
has also been detected by MS. Combining amide hydrogen exchange (HX) with MS has opened new possibilities for studying protein structure and dynamics. This article discusses models for interpreting HX results, describes studies of the structures and dynamics of small and large proteins, and summarizes the relative merits of NMR and MS for detecting H/D exchange.
Determining the locations of all atoms in a protein to within 2–3 Å (the average structure of a protein) is a milestone, because this information is intimately linked to a protein’s function. For example, with a high-resolution structure in hand, rational drug design can begin. Physical methods used to determine the three-dimensional structures of proteins can be classified according to their resolution. X-ray crystallography and high-resolution multidimensional NMR provide the highest resolution at 15 h), the equilibrium constants for each step of the unfolding process were determined directly from the populations of N, I1, I2, and U. Alternatively, the equilibrium constants were determined from the ratios of unfolding and refolding rate constants as determined by modeling. Estimated free energies for the same unfolding processes using these two approaches differed by > k–1 (Figure 1). Deconvoluting the two envelopes provided the date for determining the populations of molecules that unfolded during the labeling time (blue in Figure 4a). The rate constant for unfolding, calculated from the change in this population with time, showed that part of the SH3 domain unfolds and refolds every 15–20 min, which is far too slow to be detected by conventional HX NMR methods. This unfolding, however, is only partial. The difference in mass between the two envelopes (red and blue in Figure 4a) indicates that at least 17 residues participated in this cooperative unfolding process. The location of these unfolding residues was determined by a similar analysis of peptic fragments of labeled SH3. Two fragments displayed bimodal isotope patterns similar to those in the mass spectra of the intact protein. This bimodal isotope pattern is illustrated in Figure 4b for a peptic fragment that includes residues 44–61. Spectra for the same fragment taken from SH3 but labeled for only 3 min—before a significant population had unfolded—as well as from SH3 labeled for 45 min—after most of the SH3 had unfolded at least once—are included in Figure 4b. Mass spectra of a peptic fragment with residues 119–136 suggest that some of the residues in this segment also participate in the cooperative unfolding process. The locations of these peptic fragments are indicated by red in Figure 4c. Although not directly in the binding site, the region undergoing partial cooperative unfolding was positioned to potentially
influence the binding site conformation. Results from an additional experiment with an SH3 domain bound to a 12-aminoacid peptide showed that binding slowed the unfolding rate but did not substantially change the structure of the unfolding region (24). Further studies in which the concentration of the peptide was varied indicated that unfolding persisted even when the domain was bound to the peptide ligand. The unfolding was observed in the isolated SH3 domain and in larger constructs con-
taining the SH2 domain, suggesting that it has a role in the function of Hck (25). Unfolding of other SH3 domains has been reported, but these domains unfold on timescales faster than 1 s (30). Interactions between SH3 and SH2 domains. The properties of large, multidomain proteins are often investigated by studying the isolated structural domains of the parent protein. This approach opens the possibility that some functions may not be the same in the isolated domain because they are in larger, multidomain constructs. Extrapolating the NMR structures of isolated domains to the structures of the intact protein is of particular concern. Because HX MS can compare structures by their exchange properties, this method may be useful for extrapolating the structures of isolated domains to the intact protein structure. This approach has been used, for example, to compare the structures of isolated SH3 and SH2 domains with their structures in a larger construct (25). Structures of joint constructs SH(3+2), in which SH2 directly follows SH3 in sequence, as well as structures of intact human Hck protein in the inactive state, have been reported (31). Results of these studies showed that the structures of the SH2/SH3 domains in larger constructs are very similar to those of the isolated SH2/SH3 constructs, which is consistent with the SH2 and SH3 domains folding as individual units that contact each other only through the SH3/SH2 linker. Continuous labeling experiments were used to label isolated SH3 and SH2 domains, as well as the joint SH3/SH2 construct. Differences in deuterium levels indicated minor structural changes that had escaped detection by NMR or X-ray crystallography. Results for two peptic fragments derived from an isolated SH3 domain (closed data points) and from SH3 joined to SH2 (open data points) are presented in Figures 5a and 5b. At the shortest labeling time, the peptic fragment with residues 81–86 (the N terminus of the SH3 construct), which was derived from the joint construct, had two more deuteriums than the same fragment from an isolated SH3 domain. This observation indicates that the structures of the two constructs differ in the region of residues 81–86. The deuterium level in another segment was the same for both constructs (e.g., residues 87–99, Figure 5b), suggesting similar structures.
M A Y 1 , 2 0 0 1 / A N A LY T I C A L C H E M I S T R Y
263 A
Deuterium level
(a) 5.0
(b) 10.0
4.0
8.0
3.0
6.0
2.0
4.0
1.0
2.0
0.0 0.1
1
10
0.0
100 1000
0.1
1
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100 1000
Time (min) (c)
Isotope exchange in proteins
SH(3+2) compared with isolated SH3 & SH2 Increased HX
Decreased HX
bd bc SH3
RT-loop
bb ba be
bA aB bB aA bE bF
bC
Results for other peptic fragments are summarized in Figure 5c. The intensity of red indicates the increasing levels of deuterium in the joint construct, whereas the intensity of blue indicates the decreasing levels of deuterium. These results suggest that the structure of SH3 in the joint construct is significantly more mobile—less hydrogen bonding and with better access to the solvent—than in the isolated domain. In contrast, H/D exchange was slightly lower in the SH2 domain when part of the joint construct. These observations illustrate how HX MS can be used to determine whether the structures of isolated domains can be extrapolated to larger constructs.
SH2
bD
FIGURE 5. Deuterium levels found in peptic fragments and in SH3 and SH2 domains. The differences between SH(3+2) and isolated SH2/SH3 is color coded as increasing (red) or decreasing (blue) deuterium levels in SH(3+2) relative to the levels found in the isolated SH2 and SH3 domains. The intensity of the color indicates the amount of change. (a) The peptic fragment for residues 81–86 from the SH3 domain alone (filled points) and the SH(3+2) construct (filled points). (b) Residues 87–99 from SH3 alone (filled points) and the SH(3+2) construct (filled points). (c) Isolated SH3/SH2 versus SH3/SH2 as part of the SH(3+2) construct. (Adapted with permission from Ref. 25.)
Initially, tritium was used for detecting protein structural changes by amide HX. Although H/D exchange has been detected by UV and IR methods, NMR has become the reference technique on which other methods are judged because it can detect exchange at specific protein sites. The growing use of MS to detect H/D exchange suggests that the technique offers some advantages, including the ability to detect peptides and proteins with very high sensitivity; study partially exchanged proteins when exchange has been quenched by acid; analyze large proteins, either intact or as proteolytic fragments; and determine the intermolecular distributions of deuterium. The high sensitivity of MS allows time course studies to be completed with subnanomolar quantities of proteins. More importantly, MS can analyze proteins at submicromolar concentrations. This feature aids studies of minimally soluble proteins, as well as partially unfolded proteins. Whether studies use NMR or MS, isotope exchange is usually quenched before the analysis. Quenching in NMR experiments is typically achieved by rapidly refolding the protein to its native state, where resonance signals have been previously assigned. Although refolding slows exchange at many amide linkages, exchange at many others remains too fast to be readily detected. In MS measurements, H/D exchange is quenched by decreasing the pH and temperature, which effectively slows exchange at all peptide linkages. As a result, structural changes along the entire polypeptide chain can be detected. Adding acid and lowering the temperature to quench exchange will likely be particularly useful for protein studies of large complexes in which the protein must be isolated from a complex matrix. For example, this approach has been used to simultaneously quench isotope exchange and disassemble deuterium-labeled viral capsids (32). Both ESI and laser desorption/ionization MS have been used to analyze intact proteins with molecular masses >100,000. On the other hand, although methods for analyzing large proteins by NMR are advancing, most proteins studied this way have molecular masses
