ABSTRACT
Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.
Subject(s)
Antineoplastic Agents/administration & dosage , Fluorine/administration & dosage , Multiple Myeloma/drug therapy , Oligopeptides/administration & dosage , Proteasome Inhibitors/administration & dosage , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Proliferation/drug effects , Fluorine/pharmacokinetics , Humans , Mice , Multiple Myeloma/pathology , Proteasome Inhibitors/pharmacokinetics , Xenograft Model Antitumor AssaysSubject(s)
Antineoplastic Agents/pharmacology , Clioquinol/pharmacology , Leukemia, Experimental/drug therapy , Leukemia, Myeloid, Acute/enzymology , Multiple Myeloma/enzymology , Neoplasm Proteins/antagonists & inhibitors , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Animals , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Archaeal Proteins/antagonists & inhibitors , Asian People , Cell Line, Tumor/drug effects , Cell Line, Tumor/enzymology , Clioquinol/adverse effects , Clioquinol/therapeutic use , Copper/antagonists & inhibitors , Copper/pharmacology , Drug Screening Assays, Antitumor , Endopeptidases , Humans , Japan , K562 Cells/drug effects , K562 Cells/enzymology , K562 Cells/transplantation , Leukemia, Experimental/enzymology , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/pathology , Optic Nerve Diseases/chemically induced , Optic Nerve Diseases/ethnology , Protease Inhibitors/adverse effects , Protease Inhibitors/therapeutic use , Proteasome Endopeptidase Complex , Spinal Cord Diseases/chemically induced , Spinal Cord Diseases/ethnology , U937 Cells/drug effects , U937 Cells/enzymology , U937 Cells/transplantationABSTRACT
The global fold of maltose binding protein in complex with beta-cyclodextrin has been determined using a CNS-based torsion angle molecular dynamics protocol involving direct refinement against dipolar couplings and carbonyl chemical shift changes that occur upon alignment. The shift changes have been included as structural restraints using a new module, CANI, that has been incorporated into CNS. Force constants and timesteps have been determined that are particularly effective in structure refinement applications involving high molecular weight proteins with small to moderate numbers of NOE restraints. Solution structures of the N- and C-domains of MBP calculated with this new protocol are within approximately 2 A of the X-ray conformation.
Subject(s)
Carrier Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , beta-Cyclodextrins , Carrier Proteins/metabolism , Crystallography, X-Ray , Cyclodextrins/chemistry , Cyclodextrins/metabolism , Hydrogen Bonding , Maltose-Binding Proteins , Models, Molecular , Molecular Weight , Protein Binding , Protein Structure, Tertiary , Temperature , ThermodynamicsABSTRACT
(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.
Subject(s)
Drosophila Proteins , Insect Proteins/chemistry , src Homology Domains , Animals , Drosophila , Kinetics , Mathematical Computing , Models, Chemical , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Folding , Protein Structure, TertiaryABSTRACT
The use of a short, three-residue Cu(2+)-binding sequence, the ATCUN motif, is presented as an approach for extracting long-range distance restraints from relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N-termini of proteins and binds Cu(2+) with a very high affinity. Relaxation rates of amide protons in ATCUN-tagged protein in the presence and absence of Cu(2+) can be converted into distance restraints and used for structure refinement by using a new routine, PMAG, that has been written for the structure calculation program CNS. The utility of the approach is demonstrated with an application to ATCUN-tagged ubiquitin. Excellent agreement between measured relaxation rates and those calculated on the basis of the X-ray structure of the protein have been obtained.
Subject(s)
Copper/chemistry , Ubiquitin/chemistry , Amino Acid Motifs , Binding Sites , Humans , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Thermodynamics , Ubiquitin/metabolismABSTRACT
Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.
Subject(s)
Bacteriophage T4/enzymology , Magnetic Resonance Spectroscopy , Muramidase/chemistry , Muramidase/metabolism , Amino Acid Substitution/genetics , Bacteriophage T4/genetics , Binding Sites , Entropy , Kinetics , Ligands , Models, Molecular , Motion , Muramidase/genetics , Protein Structure, Secondary , TemperatureABSTRACT
p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.
Subject(s)
Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Allosteric Regulation , Binding Sites , DNA/chemistry , DNA/genetics , DNA/metabolism , Dimerization , Humans , Models, Biological , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Deletion/genetics , Tumor Suppressor Protein p53/geneticsSubject(s)
Carrier Proteins/chemistry , Cyclodextrins/chemistry , Magnetic Resonance Spectroscopy/methods , beta-Cyclodextrins , Amino Acids/chemistry , Carbon Isotopes , Crystallography, X-Ray , Deuterium , Macromolecular Substances , Maltose-Binding Proteins , Models, Molecular , Molecular Weight , Nitrogen Isotopes , Protein Folding , TemperatureABSTRACT
A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.
Subject(s)
Asparagine/chemistry , Bacteriophage T4/enzymology , Glycine/chemistry , Muramidase/chemistry , Mutation , Proteins/chemistry , Models, Molecular , Muramidase/genetics , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , X-Ray DiffractionABSTRACT
The measurement of residual dipolar couplings in weakly aligned proteins can potentially provide unique information on their structure and dynamics in the solution state. The challenge is to extract the information of interest from the measurements, which normally reflect a convolution of the structural and dynamic properties. We discuss here a formalism which allows a first order separation of their effects, and thus, a simultaneous extraction of structural and motional parameters from residual dipolar coupling data. We introduce some terminology, namely a generalized degree of order, which is necessary for a meaningful discussion of the effects of motion on residual dipolar coupling measurements. We also illustrate this new methodology using an extensive set of residual dipolar coupling measurements made on (15)N,(13)C-labeled human ubiquitin solvated in a dilute bicelle solution. Our results support a solution structure of ubiquitin which on average agrees well with the X-ray structure (Vijay-Kumar, et al., J. Mol. Biol. 1987, 194, 531--544) for the protein core. However, the data are also consistent with a dynamic model of ubiquitin, exhibiting variable amplitudes, and anisotropy, of internal motions. This work suggests the possibility of primary use of residual dipolar couplings in characterizing both structure and anisotropic internal motions of proteins in the solution state.
Subject(s)
Proteins/chemistry , Humans , Models, Chemical , Molecular Conformation , Protein Conformation , Solutions , Ubiquitins/chemistry , X-Ray DiffractionABSTRACT
TROSY-based HN(CO)CA 2D and 3D pulse schemes are presented for measurement of (13)C(alpha)-(13)C(beta) dipolar couplings in high molecular weight (15)N,(13)C,(2)H-labeled proteins. In one approach, (13)C(alpha)-(13)C(beta) dipolar couplings are obtained directly from the time modulation of cross-peak intensities in a set of 2D (15)N-(1)HN correlated spectra recorded in both the presence and absence of aligning media. In a second approach 3D data sets are recorded with (13)C(alpha)-(13)C(beta) couplings encoded in a frequency dimension. The utility of the experiments is demonstrated with an application to an (15)N,(13)C,(2)H-labeled sample of the ligand free form of maltose binding protein. A comparison of experimental dipolar couplings with those predicted from the X-ray structure of the apo form of this two-domain protein establishes that the relative orientation of the domains in solution and in the crystal state are very similar. This is in contrast to the situation for maltose binding protein in complex with beta-cyclodextrin where the solution structure can be generated from the crystal state via a 11 degrees domain closure.
Subject(s)
Carrier Proteins/chemistry , Cyclodextrins/chemistry , Magnetic Resonance Spectroscopy/methods , beta-Cyclodextrins , Carcinogens/chemistry , Carcinogens/metabolism , Carrier Proteins/metabolism , Cyclodextrins/metabolism , Maltose-Binding Proteins , Protein Conformation , Protein Structure, Tertiary , Radioisotopes/chemistryABSTRACT
A relaxation dispersion-based NMR experiment is presented for the measurement and quantitation of micros-ms dynamic processes at methyl side-chain positions in proteins. The experiment measures the exchange contribution to the 13C line widths of methyl groups using a constant-time CPMG scheme. The effects of cross-correlated spin relaxation between dipole-dipole and dipole-CSA interactions as well as the effects of scalar coupling responsible for mixing of magnetization modes during the course of the experiment have been investigated in detail both theoretically and through simulations. It is shown that the complex relaxation properties of the methyl spin system do not complicate extraction of accurate exchange parameters as long as care is taken to ensure that appropriate magnetization modes are interchanged in the middle of the constant-time CPMG period. An application involving the measurement of relaxation dispersion profiles of methionine residues in a Leu99Ala substitution of T4 lysozyme is presented. All of the methionine residues are sensitive to an exchange event with a rate on the order of 1200 s(-1) at 20 degrees C that may be linked to a process in which hydrophobic ligands are able to rapidly bind to the cavity that is present in this mutant.
Subject(s)
Methionine/chemistry , Muramidase/chemistry , Algorithms , Ligands , Magnetic Resonance Spectroscopy , Muramidase/genetics , MutationABSTRACT
Experiments are presented for the measurement of one-bond carbon-proton dipolar coupling values at CH and CH2 ositions in 13C-labeled, approximately 50% fractionally deuterated proteins. 13Cbeta-1Hbeta dipolar couplings have been measured for 38 of 49 possible residues in the 63-amino-acid B1 domain of peptostreptococcal protein L in two aligning media and interpreted in the context of side-chain chi1 torsion angle dynamics. The beta protons for 18 of the 25 beta-methylene-containing amino acids for which dipolar data are available can be unambiguously stereoassigned, and for those residues which are best fit to a single rotamer model the chi(1) angles obtained deviate from crystal structure values by only 5.2 degrees (rmsd). The results for 11 other residues are significantly better fit by a model that assumes jumps between the three canonical (chi1 approximately -60 degrees, 60 degrees, 180 degrees ) rotamers. Relative populations of the rotamers are determined to within +/-6% uncertainty on average and correlate with dihedral angles observed for the three molecules in the crystal asymmetric unit. Entropic penalties for quenching chi1 jumps are considered for six mobile residues thought to be involved in binding to human immunoglobulins. This study demonstrates that dipolar couplings may be used to characterize both the conformation of static residues and side-chain motion with high precision.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon Isotopes , Crystallography, X-Ray , Escherichia coli/genetics , Humans , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/metabolism , Models, Chemical , Models, Molecular , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Peptostreptococcus/chemistry , Peptostreptococcus/genetics , Protein Conformation , Protein Structure, Quaternary , Protons , ThermodynamicsABSTRACT
Maltose binding protein (MBP) is a 370-residue two-domain molecule involved in bacterial chemotaxis and sugar uptake. Rotational diffusion tensors were calculated for a complex between MBP and beta-cyclodextrin using backbone 15N T1 and T1rho relaxation times and steady state 1H-15N NOE values. The tensors obtained for each of the two domains in the protein were subsequently used to determine the relative domain orientation in the molecule. The average domain orientation determined using this approach agrees well with results from dipolar coupling data, but differs significantly from the domain orientation deduced from X-ray studies of the complex.
Subject(s)
ATP-Binding Cassette Transporters , Carrier Proteins/chemistry , Cyclodextrins/chemistry , Escherichia coli Proteins , Monosaccharide Transport Proteins , Nuclear Magnetic Resonance, Biomolecular/methods , beta-Cyclodextrins , Anisotropy , Chemical Phenomena , Chemistry, Physical , Diffusion , Escherichia coli/chemistry , Macromolecular Substances , Maltose-Binding Proteins , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RotationABSTRACT
Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented. Together with existing data on MBP in complex with beta-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and global fold that occur upon ligand binding to this two-domain protein. By measuring a large number of different one-bond residual dipolar couplings, the domain conformations, critical for biological function, were investigated for all three states of MBP. Structural models of the solution conformation of MBP in a number of different forms were generated from the experimental dipolar coupling data and X-ray crystal structures using a quasi-rigid-body domain orientation algorithm implemented in the structure calculation program CNS. Excellent agreement between relative domain orientations in ligand-free and maltotriose-bound solution conformations and the corresponding crystal structures is observed. These results are in contrast to those obtained for the MBP/beta-cyclodextrin complex where the solution state is found to be approximately 10 degrees more closed than the crystalline state. The present study highlights the utility of residual dipolar couplings for orienting protein domains or macromolecules with respect to each other.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cyclodextrins/metabolism , Escherichia coli Proteins , Escherichia coli/chemistry , Nuclear Magnetic Resonance, Biomolecular , beta-Cyclodextrins , Crystallography, X-Ray , Cyclodextrins/pharmacology , Hydrogen Bonding , Kinetics , Ligands , Models, Molecular , Periplasmic Binding Proteins , Protein Conformation/drug effects , Solutions , Trisaccharides/metabolism , Trisaccharides/pharmacologyABSTRACT
Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.
Subject(s)
Bacteriophage T4/enzymology , Muramidase/chemistry , Nuclear Magnetic Resonance, Biomolecular , Crystallography, X-Ray , Disulfides/metabolism , Models, Molecular , Muramidase/metabolism , Protein Structure, Quaternary , Protein Structure, Tertiary , Research Design , Rotation , SolutionsABSTRACT
Structural studies of proteins are critical for understanding biological processes at the molecular level. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for obtaining structural and dynamic information on proteins and protein-ligand complexes. In the present review, methodologies for NMR structure determination of proteins and macromolecular complexes are described. In addition, a number of recent advances that reduce the molecular weight limitations previously imposed on NMR studies of biomolecules are discussed, highlighting applications of these technologies to protein systems studied in our laboratories.
Subject(s)
Magnetic Resonance Spectroscopy/instrumentation , Magnetic Resonance Spectroscopy/methods , Proteins/chemistry , Models, Chemical , Models, Molecular , Protein Conformation , Spectrum Analysis/instrumentation , Spectrum Analysis/methodsABSTRACT
After more than half a century of eclipse, the mind (in contradistinction to brain and behavior) emerged in the 1950s as a legitimate object of experimental and quantitative research in natural science. This paper argues that the neural nets project of Warren S. McCulloch, in frequent collaboration with Walter Pitts, spearheaded this cognitivist turn in the 1940s. Viewing the project as a spiritual and poetic quest for the transcendental logos, as well as culturally situated epistemology, the paper focuses on McCulloch's and Pitts' efforts of logical modeling of the mind and on the social conditions that shaped that mission. From McCulloch's "experimental epistemology," the mind - purposes that ideas - emerged out of the regularities of neuronal interactions, or nets. That science of mind thus became a science of signals based on binary logic with clearly defined units of perception and precise rules of formation and transformation for representing mental states. Aimed at bridging the gulf between body and mind (matter and form) and the technical gulf between things man-made and things begotten, neural nets also laid the foundation for the field of artificial intelligence. Thus this paper also situates McCulloch;'s work within a larger historical trend, when cybernetics, information theory, systems theories, and electronic computers were coalescing into a new science of communication and control with enormous potential for industrial automation and military power in the Cold War era. McCulloch's modeling the mind as a system of command and control contributed to the actualization of this potential.
Subject(s)
Brain , Neurosciences/history , History, 20th Century , United StatesABSTRACT
The p53 tumor suppressor is a transcription factor that plays a crucial role in the activation of genes in response to DNA damage. As a first step towards detailed structural studies of the molecule aimed at understanding its regulation, we have used 4D-TROSY triple resonance NMR spectroscopy to obtain nearly complete 1H(N), 15N, 13C(alpha), 13CO and 13C(beta) resonance assignments of a dimeric form of the protein comprising DNA-binding and oligomerization domains (67 kDa). A simple comparison of 4D spectra recorded on p53 molecules consisting of DNA-binding and oligomerization domains with and without the regulatory domain establishes that both constructs have essentially identical chemical shifts. Although the affinity of p53 for target DNA is decreased in constructs containing the regulatory domain, the chemical shift results reported here suggest that this decrease is not due to specific domain interactions involving the regulatory portion of the molecule, or alternatively, that such interactions require the presence of DNA.
Subject(s)
Tumor Suppressor Protein p53/chemistry , Binding Sites , Carbon Isotopes , Carbon Monoxide , DNA/chemistry , DNA/metabolism , Dimerization , Hydrogen , Macromolecular Substances , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular/methods , Tumor Suppressor Protein p53/metabolismABSTRACT
The Leu99-->Ala mutant of T4 lysozyme contains a large internal cavity in the core of its C-terminal domain that is capable of reversibly binding small hydrophobic compounds. Although the cavity is completely buried, molecules such as benzene or xenon can exchange rapidly in and out. The dynamics of the unliganded protein have been compared to the wild-type protein by measuring the NMR spin relaxation rates of backbone amide and side chain methyl nuclei. Many residues surrounding the cavity were found to be affected by a chemical exchange process with a rate of 1500 +/- 200 s(-1), which is quenched upon addition of saturating amounts of the ligand xenon. The relationship between the structure, dynamics, and energetics of the T4 lysozyme mutant is discussed.