Papers by andrea mozzarelli
Frontiers in Molecular Biosciences, 2015
The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of ... more The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic "artifacts," including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5 ′-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms.
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1999
Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP... more Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at V s 470 nm, indicates the presence of two components characterized by lifetimes (d) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400^560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved;
Biochemistry, 1997
Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP... more Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5P-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at V s 470 nm, indicates the presence of two components characterized by lifetimes (d) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400^560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved;
Biochemistry, 1997
D-Amino acid oxidase (DAAO) is the prototype of the flavin-containing oxidases. It catalyzes the ... more D-Amino acid oxidase (DAAO) is the prototype of the flavin-containing oxidases. It catalyzes the oxidative deamination of various D-amino acids, ranging from D-Ala to D-Trp. We have carried out the X-ray analysis of reduced DAAO in complex with the reaction product imino tryptophan (iTrp) and of the covalent adduct generated by the photoinduced reaction of the flavin with 3-methyl-2-oxobutyric acid (kVal). These structures were solved by combination of 8-fold density averaging and least-squares refinement techniques. The FAD redox state of DAAO crystals was assessed by single-crystal polarized absorption microspectrophotometry. iTrp binds to the reduced enzyme with the N, C alpha, C, and C beta atoms positioned 3.8 A from the re side of the flavin. The indole side chain points away from the cofactor and is bound in the active site through a rotation of Tyr224. This residue plays a crucial role in that it adapts its conformation to the size of the active site ligand, providing the enzyme with the plasticity required for binding a broad range of substrates. The iTrp binding mode is fully consistent with the proposal, inferred from the analysis of the native DAAO structure, that substrate oxidation occurs via direct hydride transfer from the C alpha to the flavin N5 atom. In this regard, it is remarkable that, even in the presence of the bulky iTrp ligand, the active center is made solvent inaccessible by loop 216-228. This loop is thought to switch between the "closed" conformation observed in the crystal structures and an "open" state required for substrate binding and product release. Loop closure is likely to have a role in catalysis by increasing the hydrophobicity of the active site, thus making the hydride transfer reaction more effective. Binding of kVal leads to keto acid decarboxylation and formation of a covalent bond between the keto acid C alpha and the flavin N5 atoms. Formation of this acyl adduct results in a nonplanar flavin, characterized by a 22 degrees angle between the pyrimidine and benzene rings. Thus, in addition to an adaptable substrate binding site, DAAO has the ability to bind a highly distorted cofactor. This ability is relevant for the enzyme's function as a highly efficient oxidase.
Structure, 1997
Background: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules t... more Background: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong expression of a flavin-containing vanillylalcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkylphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis.
Journal of Medicinal Chemistry, 2004
Structural water molecules within protein active sites are relevant for ligand-protein recognitio... more Structural water molecules within protein active sites are relevant for ligand-protein recognition because they modify the active site geometry and contribute to binding affinity. In this work an analysis of the interactions between 23 ligands and dimeric HIV-1 protease is reported. The X-ray structures of these complexes show the presence of four types of structural water molecules: water 301 (on the symmetry axis), water 313, water 313bis, and peripheral waters. Except for water 301, these are generally complemented with a symmetry-related set. The GRID program was used both for checking water locations and for placing water molecules that appear to be missing from the complexes due to crystallographic uncertainty. Hydropathic analysis of the energetic contributions using HINT indicates a significant improvement of the correlation between HINT scores and the experimentally determined binding constants when the appropriate bridging water molecules are taken into account. In the absence of water r 2 ) 0.30 with a standard error of ( 1.30 kcal mol -1 and when the energetic contributions of the constrained waters are included r 2 ) 0.61 with a standard error of ( 0.98 kcal mol -1 . HINT was shown to be able to map quantitatively the contribution of individual structural waters to binding energy. The order of relevance for the various types of water is water 301 > water 313 > water 313bis > peripheral waters. Thus, to obtain the most reliable free energy predictions, the contributions of structural water molecules should be included. However, care must be taken to include the effects of water molecules that add information value and not just noise.
Journal of Molecular Graphics & Modelling, 2004
Getting it right" refers to the careful modeling of all elements in the living system, i.e. biolo... more Getting it right" refers to the careful modeling of all elements in the living system, i.e. biological macromolecules, ligands and water molecules. In addition, careful attention should be paid to the protonation state of ionizable functional groups on the ligands and residues at the active site. Computational technology based on the empirical HINT program is described to: (1) calculate free energy scores for ligand binding; (2) include the implicit and explicit effects of water in and around the ligand binding site; and (3) incorporate the effects of global and local pH in molecular models. This last point argues for the simultaneous consideration of a number of molecular models, each with different protonation profiles. Data from recent studies of protein-ligand systems (trypsin, thrombin, neuraminidase, HIV-1 protease and others) are used to illustrate the concepts in the paper. Also discussed are experimental factors related to accurate free energy predictions with this and other computational technologies.
Journal of Medicinal Chemistry, 2003
The prediction of the binding affinity between a protein and ligands is one of the most challengi... more The prediction of the binding affinity between a protein and ligands is one of the most challenging issues for computational biochemistry and drug discovery. While the enthalpic contribution to binding is routinely available with molecular mechanics methods, the entropic contribution is more difficult to estimate. We describe and apply a relatively simple and intuitive calculation procedure for estimating the free energy of binding for 53 protein-ligand complexes formed by 17 proteins of known three-dimensional structure and characterized by different active site polarity. HINT, a software model based on experimental LogP o/w values for small organic molecules, was used to evaluate and score all atom-atom hydropathic interactions between the protein and the ligands. These total scores (H TOTAL ), which have been previously shown to correlate with ∆G interaction for protein-protein interactions, correlate with ∆G binding for protein-ligand complexes in the present study with a standard error of (2.6 kcal mol -1 from the equation ∆G binding ) -0.001 95 H TOTAL -5.543. A more sophisticated model, utilizing categorized (by interaction class) HINT scores, produces a superior standard error of (1.8 kcal mol -1 . It is shown that within families of ligands for the same protein binding site, better models can be obtained with standard errors approaching (1.0 kcal mol -1 . Standardized methods for preparing crystallographic models for hydropathic analysis are also described. Particular attention is paid to the relationship between the ionization state of the ligands and the pH conditions under which the binding measurements are made. Sources and potential remedies of experimental and modeling errors affecting prediction of ∆G binding are discussed.
Journal of Molecular Biology, 2006
The energetics and hydrogen bonding pattern of water molecules bound to proteins were mapped by a... more The energetics and hydrogen bonding pattern of water molecules bound to proteins were mapped by analyzing structural data (resolution better than 2.3 Å ) for sets of uncomplexed and ligand-complexed proteins. Water-protein and water-ligand interactions were evaluated using hydropatic interactions (HINT), a non-Newtonian forcefield based on experimentally determined log P octanol/water values. Potential water hydrogen bonding ability was assessed by a new Rank algorithm. The HINT-derived binding energies and Ranks for second shell water molecules were K0.04 kcal mol K1 and 0.0, respectively, for first shell water molecules K0.38 kcal mol K1 and 1.6, for active site water molecules K0.45 kcal mol K1 and 2.3, for cavity water molecules K0.55 kcal mol K1 and 3.3, and for buried water molecules K0.56 kcal mol K1 and 4.4. For the last four classes, similar energies indicate that internal and external water molecules interact with protein almost equally, despite different degrees of hydrogen bonding. The binding energies and Ranks for water molecules bridging ligand-protein were K1.13 kcal mol K1 and 4.5, respectively. This energetic contribution is shared equally between protein and ligand, whereas Rank favors the protein. Lastly, by comparing the uncomplexed and complexed forms of proteins, guidelines were developed for prediction of the roles played by active site water molecules in ligand binding. A water molecule with high Rank and HINT score is unlikely to make further interactions with the ligand and is largely irrelevant to the binding process, while a water molecule with moderate Rank and high HINT score is available for ligand interaction. Water molecule displaced for steric reasons were characterized by lower Rank and HINT score. These guidelines, tested by calculating HINT score and Rank for 50 water molecules bound in the active site of four uncomplexed proteins (for which the structures of the liganded forms were also available), correctly predicted the ultimate roles (in the complex) for 76% of water molecules. Some failures were likely due to ambiguities in the structural data.
Journal of Medicinal Chemistry, 2002
The prediction of the binding affinity between a protein and ligands is one of the most challengi... more The prediction of the binding affinity between a protein and ligands is one of the most challenging issues for computational biochemistry and drug discovery. While the enthalpic contribution to binding is routinely available with molecular mechanics methods, the entropic contribution is more difficult to estimate. We describe and apply a relatively simple and intuitive calculation procedure for estimating the free energy of binding for 53 protein-ligand complexes formed by 17 proteins of known three-dimensional structure and characterized by different active site polarity. HINT, a software model based on experimental LogP o/w values for small organic molecules, was used to evaluate and score all atom-atom hydropathic interactions between the protein and the ligands. These total scores (H TOTAL ), which have been previously shown to correlate with ∆G interaction for protein-protein interactions, correlate with ∆G binding for protein-ligand complexes in the present study with a standard error of (2.6 kcal mol -1 from the equation ∆G binding ) -0.001 95 H TOTAL -5.543. A more sophisticated model, utilizing categorized (by interaction class) HINT scores, produces a superior standard error of (1.8 kcal mol -1 . It is shown that within families of ligands for the same protein binding site, better models can be obtained with standard errors approaching (1.0 kcal mol -1 . Standardized methods for preparing crystallographic models for hydropathic analysis are also described. Particular attention is paid to the relationship between the ionization state of the ligands and the pH conditions under which the binding measurements are made. Sources and potential remedies of experimental and modeling errors affecting prediction of ∆G binding are discussed.
Biophysical Journal, 2005
The unfolding and refolding kinetics of .600 single GFPmut2 molecules, entrapped in wet nanoporou... more The unfolding and refolding kinetics of .600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching or photoinduced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min ÿ1 , and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates .10 min ÿ1 , with an apparently random distribution between neutral and anionic states, that can be very different from the preunfolding equilibrium. A subsequent very slow (lifetime of ;70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules toward refolding in the original protonation state.
Journal of Molecular Biology, 1998
The T-structure of human haemoglobin is linked by salt-bridges between its four subunits, formed ... more The T-structure of human haemoglobin is linked by salt-bridges between its four subunits, formed by the C-terminal arginine residues of the a-subunits and the C-terminal histidine residues of the b-subunits. In the R-structure, these salt-bridges are absent. The oxygen af®nity of the T-structure is lower than that of the R-structure by the equivalent of 3.5 kcal/mol haem. This difference has been attributed to the constraints imposed upon the T-structure by the salt-bridges, which were thought to hinder the changes in tertiary structure needed for ®rm oxygen binding. We have subjected this postulate to a rigorous test by measuring the oxygen equilibria of T-state crystals of an abnormal human haemoglobin in which the C-terminal histidine residues of the b-chains are replaced by leucine residues. This replacement removes the salt-bridges from the histidine imidazole groups to the neighbouring aspartate residues. The crystals have an oxygen af®nity about three times greater than that of crystals of normal haemoglobin. Hill's coef®cient is close to unity. The oxygen af®nity is unaffected by pH, chloride or the allosteric effector beza®brate. Equilibrium curves determined by single crystal microspectrophometry using light polarised parallel and normal to the crystallographic a-axis show no signi®cant difference between the oxygen af®nities of a and b-haems. Our results show that rupture of salt-bridges raises the oxygen af®nity of the T-structure even when this is clamped ®rmly by the crystal lattice.
Nature, 1991
The relationship between the structure and function of haemoglobin has mainly been studied by com... more The relationship between the structure and function of haemoglobin has mainly been studied by comparing its X-ray crystal structures with its function in solutions. To make a direct comparison we have studied the functional properties of haemoglobin in single crystals, an approach that has been an important part of the investigation of several enzyme mechanisms. Here we report on the oxygen binding by single crystals of human haemoglobin grown in solutions of polyethylene glycol. Unlike haemoglobin crystals formed in concentrated salt solution, which crack and become disordered on oxygenation, crystals grown in polyethylene glycol remain intact. X-ray studies have shown that the T (deoxy) quaternary structure of haemoglobin in this crystal at pH 7.0 is maintained at atmospheric oxygen pressure, and that the salt-bridges are not broken. We find striking differences between oxygen binding by haemoglobin in this crystal and by haemoglobin in solution. Not only is oxygenation of the crystal noncooperative, but the oxygen affinity is independent of pH in the range 6.0-8.5, and is much lower than that of the T state in solution. The lack of cooperativity without a change in quaternary structure is predicted by the two-state allosteric model of Monod, Wyman and Changeux. The absence of a Bohr effect without breakage of salt-bridges is predicted by Perutz's stereochemical mechanism. In contrast to the X-ray result that oxygen binds only to the alpha haems, our measurements show that the alpha haems have only a slightly higher affinity than the beta haems.
Journal of Biological Chemistry, 2000
Proteins belonging to the superfamily of pyridoxal 5-phosphate-dependent enzymes are currently cl... more Proteins belonging to the superfamily of pyridoxal 5-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the -family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo-and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, 31 P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.
Current Medicinal Chemistry, 2007
The vitamin B(6)-derived pyridoxal 5&... more The vitamin B(6)-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and on-going research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.
Annual Review of Biophysics and Biomolecular Structure, 1996
Protein crystals contain wide solvent-filled channels that allow for traffic of metabolites and i... more Protein crystals contain wide solvent-filled channels that allow for traffic of metabolites and intramolecular motility. Ligand binding, catalysis and allosteric regulation occur in the crystalline environment but intermolecular interactions may hinder function-associated transitions and alter activity with respect to solution. Lattice constraints have, however, provided the opportunity to isolate and characterize conformational states that are poorly populated in solution. New methods are being developed to initiate reactions rapidly and synchronously throughout the crystal and to monitor their time course. A model consistent with kinetics in the crystal is necessary to interpret the results of time-resolved macromolecular crystallography.
Chemical Record, 2006
Pyridoxal 5′-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by th... more Pyridoxal 5′-phosphate (PLP)-dependent enzymes represent about 4% of the enzymes classified by the Enzyme Commission. The versatility of PLP in carrying out a large variety of reactions exploiting the electron sink effect of the pyridine ring, the conformational changes accompanying the chemical steps and stabilizing distinct catalytic intermediates, and the spectral properties of the different coenzyme-substrate derivatives signaling the reaction progress, are some of the features that have attracted our interest to investigate the structure-dynamics-function relationships of PLP-dependent enzymes. To this goal, an integrated approach combining biochemical, biophysical, computational, and molecular biology methods was used. The extensive work carried out on two enzymes, tryptophan synthase and O-acetylserine sulfhydrylase, is presented and discussed as representative of other PLP-dependent enzymes we have investigated. Finally, perspectives of PLPdependent enzymes functional genomics and drug targeting highlight the continuous novelty of an "old" class of enzymes. Scheme 4. Ketoenamine and enolimine tautomers of the external aldimine Schiff base.
Iubmb Life, 2007
We compare various allosteric models that have been proposed to explain cooperative oxygen bindin... more We compare various allosteric models that have been proposed to explain cooperative oxygen binding to hemoglobin, including the two-state allosteric model of Monod, Wyman, and Changeux (MWC), the Cooperon model of Brunori, the model of Szabo and Karplus (SK) based on the stereochemical mechanism of Perutz, the generalization of the SK model by Lee and Karplus (SKL), and the Tertiary Two-State (TTS) model of Henry, Bettati, Hofrichter and Eaton. The preponderance of experimental evidence favors the TTS model which postulates an equilibrium between high (r)- and low (t)-affinity tertiary conformations that are present in both the T and R quaternary structures. Cooperative oxygenation in this model arises from the shift of T to R, as in MWC, but with a significant population of both r and t conformations in the liganded T and in the unliganded R quaternary structures. The TTS model may be considered a combination of the SK and SKL models, and these models provide a framework for a structural interpretation of the TTS parameters. The most compelling evidence in favor of the TTS model is the nanosecond - millisecond carbon monoxide (CO) rebinding kinetics in photodissociation experiments on hemoglobin encapsulated in silica gels. The polymeric network of the gel prevents any tertiary or quaternary conformational changes on the sub-second time scale, thereby permitting the subunit conformations prior to CO photodissociation to be determined from their ligand rebinding kinetics. These experiments show that a large fraction of liganded subunits in the T quaternary structure have the same functional conformation as liganded subunits in the R quaternary structure, an experimental finding inconsistent with the MWC, Cooperon, SK, and SKL models, but readily explained by the TTS model as rebinding to r subunits in T. We propose an additional experiment to test another key prediction of the TTS model, namely that a fraction of subunits in the unliganded R quaternary structure has the same functional conformation (t) as unliganded subunits in the T quaternary structure.
Journal of Molecular Biology, 1995
To correlate directly structure with function, the oxygen affinity and the three-dimensional stru... more To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146 human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146, the COOH-terminal histidine residues of the  chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146 results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141␣ hemoglobin showed that removal of the COOH termini of the ␣ chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146 in the absence and presence of inositol hexaphosphate are 2.5-and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6-and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141␣ than those associated with His-146.
Rendiconti Lincei-scienze Fisiche E Naturali, 2006
The enormous success of structural biology challenges the physical scientist. Can biophysical stu... more The enormous success of structural biology challenges the physical scientist. Can biophysical studies provide a truly deeper understanding of how a protein works than can be obtained from staticstructures and qualitative analysis of biochemical data? We address this question in a case study by presenting the key concepts and experimental results that have led to our current understanding of cooperative oxygen binding by hemoglobin, the paradigm of structure-function relations in multi-subunit proteins. We conclude that the underlying simplicity of the two-state allosteric mechanism could not have been demonstrated without novel physical experiments and a rigorous quantitative analysis.
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Papers by andrea mozzarelli