Jonathan A. Lukin

Quaternary structure of hemoglobin in solution

Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Allosteric regulation usually involves a switch in protein conformation upon ligand binding. The energies of the corresponding structures are comparable, and, therefore, the possibility that a structure determined by x-ray diffraction in the crystalline state is influenced by its intermolecular contacts, and thus differs from the solution structure, cannot be excluded. Here, we demonstrate that the quaternary structure of tetrameric human normal adult carbonmonoxy-hemoglobin can readily be determined in solution at near-physiological conditions of pH, ionic strength, and temperature by NMR measurement of 15N-1H residual dipolar couplings in weakly oriented samples. The structure is found to be a dynamic intermediate between two previously solved crystal structures, known as the R and R2 states. Exchange broadening at the subunit interface points to a rapid equilibrium between different structures that presumably include the crystallographically observed states.

Chain-Selective Isotopic Labeling for NMR Studies of Large Multimeric Proteins: Application to Hemoglobin

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins.

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.

Molecular dynamics of HIV‐1 reverse transcriptase indicates increased flexibility upon DNA binding

HIV‐1 reverse transcriptase (RT) is one of the main targets for drugs used in the treatment of AIDS, among them, the non‐nucleoside RT inhibitors (NNRTIs). The flexibility of RT unliganded and complexed to double‐stranded DNA (RT/dsDNA), in water, has been studied by means of molecular dynamics. The simulations show that RT flexibility depends on its ligation state. The RT/dsDNA trajectories show larger fluctuations in the atomic positions than uncomplexed RT, particularly at the tips of the p66 fingers and thumb subdomains. This increased flexibility is consistent with the ability of the p66 fingers of the RT/dsDNA complex to close down after the binding of a deoxynucleoside triphosphate (dNTP) molecule, as observed in the crystal structures of RT/dsDNA bound to dNTP. The two complexation states present different patterns of concerted motions, indicating that the bound dsDNA alters RT flexibility. The motions of amino acid residues that form the non‐nucleoside RT inhibitor binding pocket upon complexation with a NNRTI are anticorrelated with the p66 fingers (in RT/dsDNA) and correlated to the RNase H subdomain (unliganded RT). These concerted motions indicate that binding of a NNRTI could alter the flexibility of the subdomains whose motions are correlated to those of the binding pocket. Proteins 2001;45:176–182.

1h, 13c, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli

Abstract1H, 13C, and 15N NMR assignments of the backbone atoms and β-carbons have been madefor liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with226 amino acid residues and a molecular weight of 24,935 Da. GlnBP is a periplasmicbinding protein which plays an essential role in the active transport of L-glutamine throughthe cytoplasmic membrane. The assignments have been obtained from three-dimensionaltriple-resonance NMR experiments on a 13C,15N uniformly labeled sample as well asspecifically labeled samples. Results from the 3D triple-resonance experiments, HNCO,HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are themain sources used to make the resonance assignments. Other 3D experiments, such asHNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirmthe resonance assignments and to extend connections where resonance peaks are missing insome of the experiments mentioned above. We have assigned more than 95% of thepolypeptide backbone resonances of GlnBP. The result of the standard manual assignment isin agreement with that predicted by an automated probabilistic method developed in ourlaboratory. A solution secondary structure of the GlnBP–Gln complex has beenproposed based on chemical shift deviations from random coil values. Eight α-helices and10 β-strands are derived using the Chemical Shift Index method.

Nuclear magnetic resonance spectroscopy in the study of hemoglobin cooperativity.

Publisher Summary Bacterial expression systems have greatly advanced NMR-based studies of Hb A and understanding of the relationship between structure and function in Hb. These advances result largely from the ability to isotopically label HbA for high-resolution heteronuclear NMR studies, literally opening up new dimensions in the NMR of hemoglobin, permitting extensive resonance assignments to be made, providing information about dynamics and hydrogen bonding, and yielding precise information regarding quaternary structure in solution. These expression systems also permit site-specific substitutions to be introduced to the Hb tetramer. NMR spectroscopy provides a powerful tool for quickly determining the effects of site-specific substitutions on the structure of the heme pocket, on site-specific distal ligand binding, on subunit interface structure and dynamics, and on the overall quaternary structure in solution.One area where NMR spectroscopy is likely to further advance the understanding of hemoglobin allostery is conformational dynamics. A few sites at the α 1 β 2 subunit interface appear to be quite dynamic over a range of time scales, and these dynamics appear to be coupled to distal ligand binding and quaternary structure. By fully characterizing the range of dynamics of different forms of Hb, NMR spectroscopy provides a picture of hemoglobin allostery that is considerably more detailed than that provided by the static structures obtained from X-ray crystallography. Another area where NMR spectroscopy is likely to advance the understanding of structure and function in hemoglobin is in determination of solution quaternary structure of the T state, using the residual dipolar coupling method. Residual dipolar coupling methods can determine whether deoxy-Hb A in solution conforms well to the crystallographically observed T-quaternary structure, or whether additional structures are also present, as in distally ligated HbCO A. Although hemoglobin has been studied with great intensity by a wide variety of methods over the last half-century, many of the molecular details that control allostery are still unknown. Experimental results on rHbs derived from NMR, equilibrium, and kinetic studies of ligand binding clearly indicate that hemoglobin is a rather flexible protein, and that its conformation can adapt to a variety of perturbations, for example, amino acid substitutions, binding of ligands, and allosteric effectors. These results also indicate that allosteric interactions in Hb A involve multiple pathways of signal transmission.