Arginine residues at internal positions in a protein are always charged

Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK_a values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK_a values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK_a; they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments.     

Large shifts in pKa values of lysine residues buried inside a protein

Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK(a) values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK(a) values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H(+) transport. The pK(a) values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK(a) values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function.     

Charges in the hydrophobic interior of proteins

Charges are inherently incompatible with hydrophobic environments. Presumably for this reason, ionizable residues are usually excluded from the hydrophobic interior of proteins and are found instead at the surface, where they can interact with bulk water. Paradoxically, ionizable groups buried in the hydrophobic interior of proteins play essential roles, especially in biological energy transduction. To examine the unusual properties of internal ionizable groups we measured the pK_a of glutamic acid residues at 25 internal positions in a stable form of staphylococcal nuclease. Two of 25 Glu residues titrated with normal pK_a near 4.5; the other 23 titrated with elevated pK_a values ranging from 5.2–9.4, with an average value of 7.7. Trp fluorescence and far-UV circular dichroism were used to monitor the effects of internal charges on conformation. These data demonstrate that although charges buried in proteins are indeed destabilizing, charged side chains can be buried readily in the hydrophobic core of stable proteins without the need for specialized structural adaptations to stabilize them, and without inducing any major conformational reorganization. The apparent dielectric effect experienced by the internal charges is considerably higher than the low dielectric constants of hydrophobic matter used to represent the protein interior in electrostatic continuum models of proteins. The high thermodynamic stability required for proteins to withstand the presence of buried charges suggests a pathway for the evolution of enzymes, and it underscores the need to mind thermodynamic stability in any strategy for engineering novel or altered enzymatic active sites in proteins.     

Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design.

Four alanine-based peptides were designed, synthesized, and tested by circular dichroism for alpha-helix formation in H2O. Each peptide has three glutamic/lysine residue pairs, is 16 or 17 amino acids long, and has blocked alpha-NH2 and alpha-COOH groups. In one set of peptides ("i+4"), the glutamic and lysine residues are spaced 4 residues or 1 residue apart. In the other set ("i+3"), the spacing is 3 or 2 residues. Within each of these sets, a pair of peptides was made in which the positions of the glutamic and lysine residues are reversed [Glu, Lys (E,K) vs. Lys, Glu (K,E)] in order to assess the interaction of the charged side chains with the helix dipole. Since the amino acid compositions of these peptides differ at most by a single alanine residue, differences in helicity are caused chiefly by the spacing and positions of the charged residues. The basic aim of this study was to test for helix stabilization by (Glu-, Lys+) ion pairs or salt bridges (H-bonded ion pairs). The results are as follows. (i) All four peptides show significant helix formation, and the stability of the alpha-helix does not depend on peptide concentration in the range studied. The best helix-former is (i+4)E,K, which shows approximately 80% helicity in 0.01 M NaCl at pH 7 and 0 degree C. (ii) The two i+4 peptides show more helix formation than the i+3 peptides. pH titration gives no evidence for helix stabilization by i+3 ion pairs. (iii) Surprisingly, the i+4 peptides form more stable helices than the i+3 peptides at extremes of pH (pH 2 and pH 12) as well as at pH 7. These results may be explained by helix stabilization through Glu-...Lys+ salt bridges at pH 7 and singly charged H bonds at pH 2 (Glu0...Lys+) and pH 12 (Glu-...Lys0). The reason why these links stabilize the alpha-helix more effectively in the i+4 than in the i+3 peptides is not known. (iv) Reversal of the positions of glutamic and lysine residues usually affects helix stability in the manner expected for interaction of these charged groups with the helix dipole. (v) alpha-Helix formation in these alanine-based peptides is enthalpy-driven, as is helix formation by the C-peptide of ribonuclease A.     

Energetics of repacking a protein interior.

To test whether interactions in the hydrophobic core of a protein can be adequately modeled based on the properties of a liquid hydrocarbon, we measured the unfolding free energies of the wild-type bacteriophage f1 gene V protein and 29 mutants with apolar substitutions at positions 35 and 47. Stability changes arising from identical mutations at these two buried sites are quite different, suggesting that one site is more rigid than the other. Reversals of residues at positions 35 and 47 confirm that their environments are distinct. Mutants containing weakly polar residues at these two sites suggest that the protein interior is more polar than a liquid hydrocarbon. Interactions between residues at the two sites appear to be minimal. These observations are compatible with a view of protein interiors that incorporates properties of liquid hydrocarbons but also includes polar interactions and a site-dependent "packing energy" associated with changes in internal structure.     

Changing hydration level in an internal cavity modulates the proton affinity of a key glutamate in cytochrome c oxidase

Cytochrome c oxidase contributes to the transmembrane proton gradient by removing two protons from the high-pH side of the membrane each time the binuclear center active site is reduced. One proton goes to the binuclear center, whereas the other is pumped to the low-pH periplasmic space. Glutamate 286 (Glu286) has been proposed to serve as a transiently deprotonated proton donor. Using unrestrained atomistic molecular dynamics simulations, we show that the size of and water distribution in the hydrophobic cavity that holds Glu286 is controlled by the protonation state of the propionic acid of heme a₃, a group on the proton outlet pathway. Protonation of the propionate disrupts hydrogen bonding to two side chains, allowing a loop to swing open. Continuum electrostatics and atomistic free-energy perturbation calculations show that the resultant changes in hydration and electrostatic interactions lower the Glu proton affinity by at least 5 kcal/mol. These changes in the internal hydration level occur in the absence of major conformational transitions and serve to stabilize needed transient intermediates in proton transport. The trigger is not the protonation of the Glu of interest, but rather the protonation of a residue ∼10 Å away. Thus, unlike local water penetration to stabilize a new charge, this finding represents a specific role for water molecules in the protein interior, mediating proton transfers and facilitating ion transport.Water is essential to the structure, dynamics, and function of biomolecules (1, 2), and its role in protein folding, association (3), and dynamics (4, 5) has been well documented. The highly polar and polarizable water molecules play diverse roles in protein interiors. Water can aid catalysis in enzyme active sites (6–8). Water or water chains are often observed in proteins that are (9, 10) proton or ion transporters or pumps (11–14). Internal cavities holding functional water molecules are believed to have a fairly constant level of hydration throughout the protein reaction cycle, unless significant conformational changes occur (15). Water penetration in response to the ionization or reduction of internal groups has been extensively discussed (16, 17), although it is usually described as part of protein''s local dielectric response.Cytochrome c oxidase (CcO) adds to the transmembrane proton gradient through proton transport coupled to electron transfer reactions (12, 18, 19). In the overall reaction, electrons from four cytochromes c are transferred to oxygen to make two water molecules at the binuclear center (BNC). The four protons needed for chemistry are bound only from the high-pH, N side of the membrane. Coupled to the process, four more protons are transferred across the membrane from the high- to low-pH (P) side of the membrane. Thus, eight charges are transferred across the membrane as each O₂ is reduced.Glu286 is a required, conserved residue that is expected to transfer protons from the D channel either to the BNC or the proton-loading site (PLS) each time CcO is reduced (Fig. 1). Experiments assign a functional pK_a to Glu286 near 9.4 (20). Thus, at higher pH, proton binding to the Glu becomes rate-limiting for steady-state turnover. The current understanding of the reaction cycle shows that protons are pumped in each of the four distinct BNC redox states (12, 18, 19). The reaction mechanism needs Glu286 to be deprotonated twice to pass a proton to the PLS and to the BNC in each CcO reduction step. Previous continuum electrostatics (21–24) and semimacroscopic (25, 26) calculations obtained pK_a values for Glu286 near 9–10. However, recent microscopic calculations have found significantly higher pK_a values of more than 12 (17, 27), making it unclear how a proton could be lost from this site, whereas others do not address the proton affinity of the essential Glu (28, 29). The discrepancy between experiment and simulations may result from technical issues such as the use of static protein structures and limited sampling of protonation states of titratable groups, or it may arise from changes in the protein that have been missed. Thus, a key question remaining is how the proton affinity of this essential Glu is modulated so it can donate a proton to the PLS and the BNC through the reaction cycle.Open in a separate windowFig. 1.Illustration of key residues near the hydrophobic cavity in CcO and general proton pathways to and from Glu286.In this work, computational studies show the hydration level of an internal cavity near Glu286 changes substantially without needing global conformational changes. Rather, the structure of an internal loop is controlled or anchored by the protonation state of the D-propionic acid of heme a₃. This potentially important motion has not been noted in previous computational studies in which part of the protein structure was constrained (21, 27, 28). Both continuum electrostatics and quantum mechanical/classical mechanical (QM/MM) free-energy simulations show that the resultant changes in Glu286 hydration level and electrostatic interactions significantly affect its pK_a (proton affinity). These findings point to a molecular mechanism to modulate the timing of proton transfers in the CcO proton pumping cycle by modifying the proton affinity of this key acid. More generally, the results show that changes in protein internal hydration may occur with only small, distal conformational changes, and these can serve as an important regulatory mechanism in ion transport, thus going beyond being part of generic dielectric response of proteins.     

Antibody specificities of children living in a malaria endemic area to inhibitory and blocking epitopes on MSP-119 of Plasmodium falciparum

Merozoite surface protein-1₁₉ (MSP-1₁₉) specific antibodies which include processing inhibitory, blocking and neutral antibodies have been identified in individuals exposed to Plasmodium falciparum. Here we intend to look at the effect of single and multiple amino acid substitutions of MSP-1₁₉ on the recognition by polyclonal antibodies from children living in Igbo-Ora, Nigeria. This would provide us with information on the possibility of eliciting mainly processing inhibitory antibodies with a recombinant MSP-1₁₉ vaccine. Blood was collected from children in the rainy season and binding of anti-MSP-1₁₉ antibodies to modified mutants of MSP-1₁₉ was analysed by ELISA. The MSP-1₁₉ mutant proteins with single substitutions at positions 22 (Leu → Arg), 43 (Glu → Leu) and 53 (Asn → Arg) and the MSP-1₁₉ mutant protein with multiple substitutions at positions 27 + 31 + 34 + 43 (Glu → Tyr, Leu → Arg, Tyr → Ser, Glu → Leu); which had inhibitory epitopes; had the highest recognition. Children recognised both sets of mutants with different age groups having different recognition levels. The percentage of malaria positive individuals (32-80%) with antibodies that bound to the mutants MSP-1₁₉ containing epitopes that recognise only processing inhibitory and not blocking antibodies, were significantly different from those with antibodies that did not bind to these mutants (21-28%). The amino acid substitutions that abolished the binding of blocking antibodies without affecting the binding of inhibitory antibodies are of particular interest in the design of MSP-1₁₉ based malaria vaccines. Although these MSP-1₁₉ mutants have not been found in natural population, their recognition by polyclonal antibodies from humans naturally infected with malaria is very promising for the future use of MSP-1₁₉ mutants in the design of a malaria vaccine.     

Identification of ionizable amino acid residues on the extracellular domain of the lutropin receptor involved in ligand binding.

The LH receptor (LHR) is a G protein-coupled receptor characterized by a relatively large N-terminal extracellular domain responsible for high affinity ligand binding. Based on a model proposed for a major portion of the extracellular domain that contains a number of leucine-rich repeats, nine ionizable amino acid residues (Glu57, Glu80, Lys158, Glu181, Lys183, Glu184, Glu188, Lys190, and Asp206) were selected for charge reversal mutagenesis based on their locations in the proposed model and their potential to serve as ligand contact sites. Mutant LHR complementary DNAs were transiently transfected into COS-7 cells, and the expressed receptors were characterized by Western blot analysis, competitive ligand (hCG) binding, and ligand-mediated cAMP production. The most interesting mutants were K158E, K183E, E184K, and D206K, which were present on the plasma membrane fraction, as judged by Western blots, but were incapable of binding hCG and, of course, were deficient in hCG-mediated cAMP production. Other replacements at these positions, K158R,Q,G; K183R,Q,G; E184N; and D206E,Q, led to cell surface binding and signaling. The mutants E57K, E189K, and K190E behaved similarly to wild-type LHR; E80K was trapped intracellularly, but bound ligand in solubilized cells; and E181K was not expressed or was rapidly degraded. These results, based on 18 point mutants of LHR, indicate that Lys158, Lys183, Glu184, and Asp206 are involved, either directly or indirectly, in gonadotropin binding and support the general nature of the proposed model.     

Conformational effects of substituting amino acids for glutamine-61 on the central transforming region of the P21 proteins.

The low-energy conformations for a series of peptides based on the sequence of the ras P21 protein from position 55 to position 67 have been computed using conformational energy analysis. These sequences differed at position 61 and contained Gln, Pro, Leu, Lys, and Arg at this position. P21 proteins with Gln, Glu, or Pro at this position do not cause cell transformation at normal levels of expression; proteins with substitutions of at least 14 other amino acids at this position (Leu, Lys, and Arg having been found in tumors in place of the normally occurring Gln-61) do cause malignant transformation of cells in culture. We find that the segments of residues 55-67 from the nontransforming proteins (Gln- or Pro-61) adopt a structure that is energetically unfavorable for the same segment with Leu, Lys, or Arg at position 61. The critical feature of this structure is an alpha-helix from residues 62 to 68. Residue 61 (Gln or Pro) adopts an extended conformation. On the other hand, the segment from transforming proteins can adopt two structures, one all alpha-helical from residue 61 to residue 68 and the other a less-regular, higher-energy structure. The segments from the normal protein can adopt the all alpha-helical structure, a finding that can explain the fact that elevated intracellular levels of the normal protein also cause cell transformation. The results of the calculations suggest that specific changes in the structure of this region can account for the oncogenic effect of the proteins in which substitutions occur.     

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pK_a values near 7. When buried together their pK_a values appear to be normal. The ionizable moieties of the buried Glu–Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu–Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu–Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.Paired ionizable groups buried in hydrophobic environments inside proteins are an uncommon but essential structural motif necessary for H⁺ transport (1), e⁻ transfer (2), catalysis (3–5), and other important biochemical processes. Despite their importance, the properties of these buried ionizable pairs are poorly understood because they are difficult to study experimentally in the proteins where they play functional roles. When the pair consists of a basic and an acidic group it is usually assumed to be charged, but this is often just speculation without direct experimental support (6, 7). In principle, a Coulomb interaction between two ionizable groups of opposite polarity buried in a dehydrated environment inside a protein can be very strong, but this has not been established experimentally. Simulations suggest that buried ion pairs are always destabilizing (8); however, from an experimental perspective it is not known if the favorable Coulomb interaction would be sufficient to compensate for the unfavorable dehydration of the two buried charges or if the net effect of the pair on the thermodynamic stability of a protein would be stabilizing or not. These issues were examined in detail with an artificial ion pair buried in the hydrophobic interior of staphylococcal nuclease (SNase).The buried ion pair was engineered by introducing the V23E and L36K substitutions in a highly stable form of SNase. The pK_a values of Glu-23 and Lys-36 introduced individually are anomalous (pK_a of 7.1 for Glu-23, ref. 9; and 7.2 for Lys-36, ref. 10), consistent with the groups existing in hydrophobic environments that are neither as polar nor as polarizable as water (11–13). Structural modeling indicated that the ionizable moieties of Glu-23 and Lys-36 could form a short-range interaction when buried simultaneously. Their similar pK_a values were expected to facilitate H⁺ transfer between the acidic and basic moieties. The main goals of this study were to determine the charge state of an internal pair of Glu–Lys residues, to examine how such a pair could affect its microenvironment, to measure consequences on thermodynamic stability of the protein, and by comparing the pair against the single-site substitutions, to evaluate the relative magnitudes of Coulomb interactions and dehydration energies.According to primitive, macroscopic electrostatics models, the transfer of an ion pair from water into a cavity with low dielectric constant is always unfavorable (Fig. 1). In these models the favorable Coulomb interaction between the charges in the buried pair is never strong enough to offset the unfavorable self-energy experienced by the two buried charges. In some microscopic models, Coulomb interactions can be stronger than dehydration effects, but calculations with these methods are still suspect as they cannot reproduce self-energies reliably (14, 15) and tend to overestimate the magnitude of Coulomb interactions (14, 16–18). The experiments that were performed test assumptions made in most continuum methods, including the idea that a pair of ionizable groups interacting at close range in a dehydrated environment can be treated classically as point charges without invoking some level of quantum mechanical treatment to account for polarizability and for the properties of a proton shared between acidic and basic moieties interacting over a short distance in a highly dehydrated environment (19, 20).Open in a separate windowFig. 1.Free energy for transfer of a charged pair from water into a dielectric cavity of variable dielectric constant ε_prot, calculated with a primitive continuum electrostatics model and dissected into contributions from dehydration and Coulomb energies. Dehydration energy (red) calculated with a Born formalism to describe the energy for the transfer of two charges of opposite sign, each of radius r_ion = 1.75 Å, from water into the center of the spherical dielectric cavity of radius r_cavity = 20 Å. Coulomb interaction (blue) between the two charges separated by a distance r_ij = 3.5 Å, calculated with Coulomb’s law relative to the Coulomb energy in water (ε_prot = 80). Total transfer free energy (black) for moving the ion pair from water (ε_w = 80) into the center of the cavity, is calculated as a function of ε_prot, the dielectric constant of the cavity.Improved computational methods for structure-based electrostatics calculations are necessary for progress in molecular understanding of catalysis and other biochemical processes governed by acid–base chemistry. The physical insight gained from the present study will help guide the development of more accurate structure-based calculations. The thermodynamic and structural data from this study will also constitute benchmarks useful for stringent testing and validation of structure-based calculations. Our results suggest a strategy for the evolution or engineering of enzymatic active sites capable of performing acid–base chemistry and proton pumping focused more on the use of a protein scaffold with high thermodynamic stability than on the manipulation of the microenvironments and chemical details needed to stabilize buried charge clusters.     

Functional segregation of the highly conserved basic motifs within the third endoloop of the human secretin receptor

In this study, a mutagenesis-based strategy was employed to assess the roles of two highly conserved motifs (KLR and RLAR) within the third endoloop of the human secretin receptor. Block deletion of KLRT and mutation of Lys323 (K(323)I) significantly reduced cAMP accumulation, and these mutations did not affect ligand interaction and receptor number expressed on the cell surface. Thus, the KLRT region at the N terminus of the third endoloop, particularly Lys323, is important for G protein coupling. For the RLAR motif, receptors with substitutions at positions 339 and 342 from Arg to Ala (R(339, 342)A), Glu (R(339, 342)E), or Ile (R(339, 342)I) as well as block deletion of the RLAR motif were all found to be defective in both secretin-binding and cAMP production. Interestingly, a single mutation at the corresponding positions of Arg339 or Arg342 responded as the wild-type human secretin receptor in all functional assays, indicating that the presence of one Arg at either position within the RLAR motif is sufficient for a normal receptor function. Immunofluorescent staining of these mutant receptors showed that these Arg residues are responsible for surface presentation and/or receptor stability.     

Interactions between amino acid side chains in cylindrical hydrophobic nanopores with applications to peptide stability

Confinement effects on protein stability are relevant in a number of biological applications ranging from encapsulation in the cylindrical cavity of a chaperonin, translocation through pores, and structure formation in the exit tunnel of the ribosome. Consequently, free energies of interaction between amino acid side chains in restricted spaces can provide insights into factors that control protein stability in nanopores. Using all-atom molecular dynamics simulations, we show that 3 pair interactions between side chains—hydrophobic (Ala–Phe), polar (Ser–Asn) and charged (Lys–Glu)—are substantially altered in hydrophobic, water-filled nanopores, relative to bulk water. When the pore holds water at bulk density, the hydrophobic pair is strongly destabilized and is driven to large separations corresponding to the width and the length of the cylindrical pore. As the water density is reduced, the preference of Ala and Phe to be at the boundary decreases, and the contact pair is preferred. A model that accounts for the volume accessible to Phe and Ala in the solvent-depleted region near the pore boundary explains the simulation results. In the pore, the hydrogen-bonded interactions between Ser and Asn have an enhanced dependence on their relative orientations, as compared with bulk water. When the side chains of Lys and Glu are restrained to be side by side, parallel to each other, then salt bridge formation is promoted in the nanopore. Based on these results, we argue and demonstrate that for a generic amphiphilic sequence, cylindrical confinement is likely to enhance thermodynamic stability relative to the bulk.     

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the "peroxy" state, P(r)) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH approximately 7.5. The movement of the Lys is proposed to regulate proton transfer by "shutting off" the protonic connectivity through the K-pathway after initiation of the O(2) reduction chemistry. This "shutoff" prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.     

Six point mutations that cause factor XI deficiency

We have identified six novel types of mutation that cause factor XI deficiency, an inherited bleeding disorder. Two are point mutations that interfere with the normal splicing of exons in the mRNA and four are point mutations that result in amino acid substitutions. One of these amino acid substitutions (Asp 16-->His) is near the amino terminal end of the protein. The other three amino acid substitutions (Leu 302-->Pro, Thr 304-->Ile, and Glu 323-->Lys) are in the fourth apple domain, a region that mediates dimerization of identical subunits of factor XI. All four amino acid substitutions cause a reduction in the amount of factor XI secreted from cells grown in vitro.     

Prion protein NMR structure and species barrier for prion diseases

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein–protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.     

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer-dimer interface of the tetrameric oligomerization domain of p53 (residues 325-355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the alpha-helices at the dimer-dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.     

Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium channel

Structural models of inward rectifier K⁺ channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as α helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in K_ir2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in Xenopus oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with α helices or β strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.     

Interaction between cytochrome b5 and hemoglobin: involvement of beta 66 (E10) and beta 95 (FG2) lysyl residues of hemoglobin.

In erythrocytes the reduction of oxidized hemoglobin (methemoglobin) is dependent upon an electron transport reaction between cytochrome b5 and methemoglobin. These two proteins are believed to form a complex whose bonding is principally determined by complementary charge interactions between acidic groups of cytochrome b5 and basic groups of hemoglobin. In order to refine this model, three surface lysyl hemoglobin variants--namely Hb N Baltimore beta 95 (FG2) Lys leads to Glu, Hb I Toulouse beta 66 (E10) Lys leads to Glu, and Hb I Philadelphia alpha 16 (A14) Lys leads to Glu--have been studied with respect to their reducibility and ability to bind cytochrome b5. In the two former variants, the substituted amino acids are located near the heme crevice; in the third one the substitution lies far from it. Substitutions of lysine for glutamic acid in positions beta 66 and beta 95 perturb the formation of the cytochrome b5--hemoglobin complex and result in a dramatic impairment of the cytochrome b5-mediated reduction, whereas the same mutation in position alpha 16 has no effect. We conclude that the lysine residues in positions beta 66 and beta 95 are directly involved in the binding of cytochrome b5. The three-dimensional structure of hemoglobin suggests that the cytochrome b5-binding domain of hemoglobin is constituted by four lysine residues surrounding the heme crevice in both alpha and beta chains. Similarities with other interacting hemoproteins are discussed.