Backbone Amide Hydrogen Exchange Research Articles

Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein

It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins.

Hydrogen Exchange Reveals the Mechanism of Stabilization of P53 Rescue Mutants N235K and N239Y

p53 protein is a transcription factor important for regulating DNA repair and cellular apoptosis. It has been shown that the dysfunction of p53 protein is sufficient to allow for uncontrolled cell proliferation. Notably, 50% of all cancers have a mutated form of p53, most of these are simple missense mutations localized in the DNA binding domain (DBD). Previous studies have shown that the function of p53 can be restored by introducing secondary mutations known as suppressor or rescue mutations. Specifically, a cancer suppressor mutation motif consisting of residues 235-239-240 has been shown to rescue 16 common cancer mutations that are known to unfold the DBD. The individual rescue mutation N239Y is known to increase thermal stability. In contrast, the stability of N235K is similar to WT, leaving the mechanism of rescue undefined. Notably, the crystal structures of these two rescue mutants are virtually identical to the WT conformation; thus, the details of how these changes compensate for the destabilization of cancer mutants is not obvious on inspection of the static structure. We set out to gain further insight about the mechanism by which both of these mutations restore function to p53 cancer mutants using backbone amide hydrogen exchange measured by NMR spectroscopy. Protection factor analysis shows that specific residues throughout the beta-sheet scaffold increase in both mutants compared to the WT p53 revealing distinct changes in dynamics. Both rescue mutations alter the profile of positions that are most protected from exchange and, consequently, show increased stability. These data provide a model for stabilizing effects that could serve as a metric in evaluating anti-cancer therapeutics that target p53.

Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data

The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. Graphical Abstract ᅟ.

Determination of Equine Cytochrome c Backbone Amide Hydrogen/Deuterium Exchange Rates by Mass Spectrometry Using a Wider Time Window and Isotope Envelope.

A new strategy to analyze amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data is proposed, utilizing a wider time window and isotope envelope analysis of each peptide. While most current scientific reports present HDX-MS data as a set of time-dependent deuteration levels of peptides, the ideal HDX-MS data presentation is a complete set of backbone amide hydrogen exchange rates. The ideal data set can provide single amide resolution, coverage of all exchange events, and the open/close ratio of each amide hydrogen in EX2 mechanism. Toward this goal, a typical HDX-MS protocol was modified in two aspects: measurement of a wider time window in HDX-MS experiments and deconvolution of isotope envelope of each peptide. Measurement of a wider time window enabled the observation of deuterium incorporation of most backbone amide hydrogens. Analysis of the isotope envelope instead of centroid value provides the deuterium distribution instead of the sum of deuteration levels in each peptide. A one-step, global-fitting algorithm optimized exchange rate and deuterium retention during the analysis of each amide hydrogen by fitting the deuterated isotope envelopes at all time points of all peptides in a region. Application of this strategy to cytochrome c yielded 97 out of 100 amide hydrogen exchange rates. A set of exchange rates determined by this approach is more appropriate for a patent or regulatory filing of a biopharmaceutical than a set of peptide deuteration levels obtained by a typical protocol. A wider time window of this method also eliminates false negatives in protein-ligand binding site identification. Graphical Abstract ᅟ.

Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10-30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1-3 h). Here various experimental conditions, as well as a quenched-flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044-1 000 000 s), when converted into the standard exchange condition (pH 7 and 23°C).

Gas-Phase Fragmentation of Peptides by MALDI in-Source Decay with Limited Amide Hydrogen (1H/2H) Scrambling

To achieve a fundamental understanding of the function of proteins and protein complexes at the molecular level, it is crucial to obtain a detailed knowledge about their dynamic and structural properties. The kinetics of backbone amide hydrogen exchange is intimately linked to the structural dynamics of the protein, and in recent years, the monitoring of the isotopic exchange of these hydrogens by mass spectrometry has become a recognized method. At present, the resolution of this method is, however, limited and single-residue resolution is typically only obtained for a few residues in a protein. It would therefore be desirable if gas-phase fragmentation could be used to localize incorporated deuterons as this would ultimately lead to single-residue resolution. A central obstacle for this approach is, however, the occurrence of intramolecular migration of amide hydrogens upon activation of the gaseous protein (i.e., hydrogen scrambling). Here we investigate the occurrence of scrambling in selectively labeled peptides upon fragmentation by matrix-assisted laser desorption/ionization in-source decay (MALDI ISD). We have utilized peptides with a unique regioselective deuterium incorporation that allows us to accurately determine the extent of scrambling upon fragmentation. Our results show that the level of scrambling upon MALDI ISD is so low that the solution deuteration pattern is readily apparent in the gas-phase fragment ions. These results suggest that MALDI ISD may prove useful for hydrogen exchange studies of purified peptides and small proteins.

Post-translational modification of barley LTP1b: The lipid adduct lies in the hydrophobic cavity and alters the protein dynamics

NMR techniques have been used to characterise the effects of a lipid-like post-translational modification on barley lipid transfer protein (LTP1b). NMR chemical shift data indicate that the lipid-like molecule lies in the hydrophobic cavity of LTP1b, with Tyr 79 being displaced to accommodate the ligand in the cavity. The modified protein has a reduced level of backbone amide hydrogen exchange protection, presumably reflecting increased dynamics in the protein. This may result from a loosening of the protein structure and may explain the enhanced surface properties observed for LTP1b.

Structural and Dynamic Perturbations Induced by Heme Binding in Cytochrome b5,

The water-soluble domain of rat hepatic cytochrome b(5) undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b(5) shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.

The native state of apomyoglobin described by proton NMR spectroscopy: the A-B-G-H interface of wild-type sperm whale apomyoglobin.

Proton nuclear magnetic resonance spectroscopy was applied to sperm whale apomyoglobin to describe the conformation adopted by the protein under native conditions. The study focused on the A-B-G-H interface, a region known to form a compact subdomain in the apoprotein (Hughson and Baldwin, Biochemistry 28:4415-4422, 1989). Two histidine residues located in this subdomain, His24 and His119, interact and are thought to play a role in the acid denaturation process (Barrick et al., J. Mol. Biol. 237:588-601, 1994). A stable double mutant at these positions (His24Val/His119Phe sperm whale apomyoglobin) was compared with wild-type apomyoglobin. The amino acid replacements result in chemical shift perturbations near the mutations, in particular in the AB interhelical region, and in a deceleration of backbone amide hydrogen exchange in the B helix from position 27 to position 33. The double mutant data were used to expand and confirm the wild-type spectral analysis. Signals from the D helix were identified that demonstrate the formation of holoprotein-like structure. The assigned wild-type nuclear Overhauser effects, although in small number, were sufficient to construct a model of the compact subdomain of the apoprotein. This was achieved by using the structure of the holoprotein and restraining it with the geometrical information on the apoprotein in a simulated annealing procedure. The experimental restraints define a low-resolution model of the A-B-G-H interface in apomyoglobin.

Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements.

The backbone motions of calcium-free Xenopus calmodulin have been characterized by measurements of the 15N longitudinal relaxation times (T1) at 51 and 61 MHz, and by conducting transverse relaxation (T2), spin-locked transverse relaxation (T1 rho), and 15N-[1H] heteronuclear NOE measurements at 61 MHz 15N frequency. Although backbone amide hydrogen exchange experiments indicate that the N-terminal domain is more stable than calmodulin's C-terminal half, slowly exchanging backbone amide protons are found in all eight alpha-helices and in three of the four short beta-strands. This confirms that the calcium-free form consists of stable secondary structure and does not adopt a 'molten globule' type of structure. However, the C-terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 microseconds, which affects many of the C-terminal domain residues. This results in significant shortening of the 15N T2 values relative to T1 rho, whereas the T1 rho and T2 values are of similar magnitude in the N-terminal half of the protein. A model in which the motion of the protein is assumed to be isotropic suggests a rotational correlation time for the protein of about 8 ns but quantitatively does not agree with the magnetic field dependence of the T1 values and does not explain the different T2 values found for different alpha-helices in the N-terminal domain. These latter parameters are compatible with a flexible dumb-bell model in which each of calmodulin's two domains freely diffuse in a cone with a semi-angle of about 30 degrees and a time constant of about 3 ns, whereas the overall rotation of the protein occurs on a much slower time scale of about 12 ns. The difference in the transverse relaxation rates observed between the amides in helices C and D suggests that the change in interhelical angle upon calcium binding is less than predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. & James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1-17].

Characterization of an independent structural unit in apocytochrome b5

Apocytochrome b5 is a partially folded protein which contains a stable structural unit under native conditions [Moore, C.D., Al-Misky, O.N., & Lecomte, J.T.J. (1990) Biochemistry 30, 8357-8365]. In this work, the fold of the unit was examined by using 1H and 15N-edited two-dimensional NMR spectroscopy. It was found that it contains four of the five beta-strands and two of the six alpha-helices present in the holoprotein. The remainder of the structure appears to be mostly unstructured and fluctuating among several conformations. The structural unit is stabilized by a hydrophobic core formed by residues from each of the folded elements of secondary structure. Nuclear Overhauser effects and chemical shift values demonstrated that the unit is structurally similar in the apo- and holoproteins. However, the backbone amide hydrogen exchange was found to be much accelerated in the apoprotein. The paramagnetic relaxation agent HyTEMPO was used to probe the packing of the structure. HyTEMPO has unrestricted access to the empty heme binding site whereas it is unable to penetrate the stabilizing core. It was concluded that addition of the heme is necessary for the last strand to dock properly to the rest of the sheet. The kinetics of refolding of the apoprotein were monitored by stopped-flow fluorescence spectroscopy. Extensive protection of the sole tryptophan residue by docking of the two polypeptide termini occurs in less than 60 ms. It was proposed that apocytochrome b5, with its two-region behavior, might serve as a model for the design of proteins which bind a prosthetic group.

NMR studies of the influence of dodecyl sulfate on the amide hydrogen exchange kinetics of a micelle-solubilized hydrophobic tripeptide.

Backbone amide hydrogen exchange measurements are an important source of information about the internal dynamics of proteins. Before such measurements can be interpreted unambiguously, contributions to hydrogen exchange rates from the chemical and physical environment of the amides must be taken into account. Membrane proteins are often solubilized in detergents, yet there have not been any systematic investigations of the possible effects detergents may have on the amide hydrogen exchange rates of proteins. To address this question, we have measured individual backbone and carboxyl-terminal amide exchange rates for the amphipathic tripeptide Leu-Val-Ile-amide dissolved in water and dodecyl sulfate micelles. 1H NMR spectroscopy was used to measure exchange using the direct exchange-out into D2O technique at 5 degrees C and using an indirect steady-state saturation-transfer technique at 25 degrees C. The broadening effect of micelle-incorporated spin-labeled fatty acid (12-doxylstearate) on the 1H NMR spectra of both the detergent and the peptide resonances was used to demonstrate that the tripeptide is intimately associated with the micelle. The resonance from formate ion, which is excluded from the micelle, was unperturbed by the spin label. The detergent did not retard the exchange rates of either the primary (terminal) or secondary (backbone) amides of the tripeptide. This suggests that the micelle/peptide interaction does not restrict access of charged catalysts and water to these amides and shows that the peptide amides are not hydrogen bonded. However, the pH for the exchange minima of these amides in detergent was increased between 1.2 and 1.7 units compared to exchange in water.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by 1H NMR spectroscopy.

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. We have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using 1H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D2O at 24 degrees C were used to follow the exchange of the slowest amides in the protein. Multiple exponential fitting of the exchange data showed that these amides (29 +/- 3 at pH 4.5) exchanged in two kinetic sets with exchange rates [(1.2 +/- 0.4) x 10(-4) s-1 and (4.1 +/- 1.2) x 10(-7) s-1] that differed by more than 100-fold, the slower kinetic set being retarded 10(5)-fold relative to poly(DL-alanine). The exchange rate constant for the slowest set of amides exhibited an unusual pD dependence, being proportional to [OD-]1/2. It is shown that this is an artifact of the multiple exponential fitting of the data, and a new method of presentation of exchange data as a function of pD is introduced. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55 degrees C and that about 30 amides have exchange rates retarded by at least 10(5)-fold at 24 degrees C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. This is explained as being due to long-range electrostatic effects arising both from the protein itself and also from the anionic detergent molecules. Hydrogen exchange studies on the products of proteinase K digestion of the protein localized the slowly exchanging amides to the hydrophobic core of the protein. Relaxation [Henry, G.D., Weiner, J.H., & Sykes, B.D. (1986) Biochemistry 25, 590-598] and solid-state NMR experiments [Leo, G.C., Colnago, L.A., Valentine, K.G., & Opella, S.J. (1987) Biochemistry 26, 854-862] have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile.(ABSTRACT TRUNCATED AT 400 WORDS)