Characterizing semilocal motions in proteins by NMR relaxation studies

The understanding of protein function is incomplete without the study of protein dynamics. NMR spectroscopy is valuable for probing nanosecond and picosecond dynamics via relaxation studies. The use of ¹⁵N relaxation to study backbone dynamics has become virtually standard. Here, we propose to measure the relaxation of additional nuclei on each peptide plane allowing for the observation of anisotropic local motions. This allows the nature of local motions to be characterized in proteins. As an example, semilocal rotational motion was detected for part of a helix of the protein Escherichia coli flavodoxin.     

Age-dependent deamidation of asparagine residues in proteins

Nonenzymatic deamidation of peptides and proteins represents an important degradation reaction occurring in vitro in the course of isolation or storage and in vivo during development and/or aging of cells. This review first presents a synopsis of the influence of structure on deamidation reaction proceeding via a five-membered succinimide intermediate, followed by an outline of procedures for separation and detection of deamidated forms. Selected examples for in vitro and in vivo deamidation are reviewed including the possible biological consequences of this protein degradation. Finally, the reaction of protein methyltransferase with L-isoaspartyl- and D-aspartyl residues and its possible role in protein repair is elucidated.     

Low-frequency normal mode in DNA HhaI methyltransferase and motions of residues involved in the base flipping

The results of normal-mode analyses are in accord with the proposal that a low-frequency motion of the HhaI methyltransferase enzyme is responsible for base flipping in bound DNA. The vectors of the low-frequency normal mode of residues Ser-85 and Ile-86 point directly to the phosphate and ribose moieties of the DNA backbone near the target base in position to rotate the dihedral angles and flip the base out of the DNA duplex. The vector of residue Gln-237 on the major groove is in the proper orientation to assist base separation. Our results favor the major groove pathway and the protein active process in base flipping.     

Long-range electron exchange measured in proteins by quenching of tryptophan phosphorescence.

Ten proteins that span a wide range of phosphorescence lifetimes were examined for sensitivity to quenching by four agents of disparate chemical nature. The results show that quenching efficiency is relatively independent of the quencher and is highly correlated with depth of burial of the phosphorescent tryptophan. The bimolecular quenching rate constants (kq) measured for the different proteins, spanning 5 orders of magnitude in kq, are found to decrease exponentially with the distance (r) of the tryptophan in angstroms from the protein surface--i.e., kq = Aexp(-r/rho), where A contains a geometrical factor dependent on tryptophan burial and surface geometry [corrected]. Theoretical analysis shows that this behavior can be expected for an electron-exchange reaction between the buried tryptophans and quenchers in solution in the rapid diffusion limit. Therefore, the results obtained provide evidence for an exponential dependence of electron-transfer rate on distance in a protein environment and evaluate the distance parameter, rho, for electron transfer through the general protein matrix at 1.0 A. For a unimolecular donor-acceptor pair with ket = koexp(-r/rho), ko approximately 10(9) sec-1.     

Oxidation of methionine residues in proteins of activated human neutrophils.

A simple assay for the detection of 35S-labeled methionine sulfoxide residues in proteins is described. The assay, which is based on the ability of CNBr to react with methionine but not with methionine sulfoxide, requires the prelabeling of cellular proteins with [35S]methionine. The assay was used to study the extent of methionine oxidation in newly synthesized proteins of both activated and quiescent human neutrophils. In cells undergoing a phorbol 12-myristate 13-acetate-induced respiratory burst, about 66% of all methionine residues in newly synthesized proteins were oxidized to the sulfoxide derivative, as compared with 9% in cells not treated with the phorbol ester. In contrast, quantitation of methionine sulfoxide content in the total cellular protein by means of amino acid analysis showed that only 22% of all methionine residues were oxidized in activated cells as compared with 9% in quiescent cells. It is proposed that methionine residues in nascent polypeptide chains are more susceptible to oxidation than those in completed proteins.     

Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings

Despite their importance for biological activity, slower molecular motions beyond the nanosecond range remain poorly understood. We have assembled an unprecedented set of experimental NMR data, comprising up to 27 residual dipolar couplings per amino acid, to define the nature and amplitude of backbone motion in protein G using the Gaussian axial fluctuation model in three dimensions. Slower motions occur in the loops, and in the beta-sheet, and are absent in other regions of the molecule, including the alpha-helix. In the beta-sheet an alternating pattern of dynamics along the peptide sequence is found to form a long-range network of slow motion in the form of a standing wave extending across the beta-sheet, resulting in maximal conformational sampling at the interaction site. The alternating nodes along the sequence match the alternation of strongly hydrophobic side chains buried in the protein core. Confirmation of the motion is provided through extensive cross-validation and by independent hydrogen-bond scalar coupling analysis that shows this motion to be correlated. These observations strongly suggest that dynamical information can be transmitted across hydrogen bonds and have important implications for understanding collective motions and long-range information transfer in proteins.     

High tolerance for ionizable residues in the hydrophobic interior of proteins

Internal ionizable groups are quite rare in water-soluble globular proteins. Presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. Here we show that proteins can have an inherently high tolerance for internal ionizable groups. The 25 internal positions in staphylococcal nuclease were substituted one at a time with Lys, Glu, or Asp without abolishing enzymatic activity and without detectable changes in the conformation of the protein. Similar results with substitutions of 6 randomly chosen internal positions in ribonuclease H with Lys and Glu suggest that the ability of proteins to tolerate internal ionizable groups might be a property common to many proteins. Eighty-six of the 87 substitutions made were destabilizing, but in all but one case the proteins remained in the native state at neutral pH. By comparing the stability of each variant protein at two different pH values it was established that the pK_a values of most of the internal ionizable groups are shifted; many of the internal ionizable groups are probably neutral at physiological pH values. These studies demonstrate that special structural adaptations are not needed for ionizable groups to exist stably in the hydrophobic interior of proteins. The studies suggest that enzymes and other proteins that use internal ionizable groups for functional purposes could have evolved through the random accumulation of mutations that introduced ionizable groups at internal positions, followed by evolutionary adaptation and optimization to modulate stability, dynamics, and other factors necessary for function.     

Structural changes involved in protein binding correlate with intrinsic motions of proteins in the unbound state

Protein-protein binding usually involves structural changes that may extend beyond the rearrangements on a local scale, and cannot be explained by a classical lock-and-key mechanism. Several models have been advanced to explain the flexible binding of proteins such as the induced fit mechanism where the ligand is postulated to induce a conformational change at the interaction site upon binding, or the preexisting equilibrium hypothesis that assumes that protein samples an ensemble of conformations at equilibrium conditions and that the ligand binds selectively to an active conformation. We explored the equilibrium motions of proteins that exhibit relatively large (nonlocal) conformational changes upon protein binding using the Gaussian network model and the anisotropic network model of protein dynamics. For four complexes, LIR-1/HLA-A2, Actin/DNase I, CDK2/cyclin, and CDK6/p16(INK4a), the motions calculated for the monomer exhibiting the largest conformational change, in its unbound (free) form, correlate with the experimentally observed structural changes upon binding. This study emphasizes the preexisting equilibrium/conformational selection as a mechanism for protein-protein interaction and lends support the concept that proteins, in their native conformation, are predisposed to undergo conformational fluctuations that are relevant to, or even required for, their biological functions.     

Modification of histidine residues in proteins by reaction with 4-hydroxynonenal.

We find that histidine residues in proteins are major targets for reaction with the lipid peroxidation product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivatives upon subsequent treatment with NaB[3H]H4. Amino acid analysis of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stiochiometric formation of 3H-labeled amino acid derivatives. The same labeled products were detected in acid hydrolysates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the production of two compounds. Upon acid hydrolysis, both derivatives yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of N pi (N tau)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha, beta-unsaturated bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be determined.     

Structure of human hemopexin: O-glycosyl and N-glycosyl sites and unusual clustering of tryptophan residues.

The primary structure of human hemopexin is being deduced from sequence analysis of a series of peptides obtained from chemical and enzymatic digests of the protein. Human hemopexin consists of about 440 amino acid residues. It has five sites of attachment of glucosamine oligosaccharides at the signal sequence of Asn-X-Thr/Ser. A unique structural feature is the virtual blocking of the amino-terminal threonine residue, which is O-linked to a galactosamine oligosaccharide that has not previously been identified in this protein. The galactosamine oligosaccharide and one glucosamine oligosaccharide are located in the amino-terminal region, three of the glucosamine oligosaccharides are in the middle region, and one glucosamine oligosaccharide is in the carboxyl-terminal region of the protein. Two of the five glucosamine oligosaccharides are present in a histidine-rich sequence of the middle region of the protein, in which the histidines flank beta-turns presumably at the surface of hemopexin. Clusters of tryptophan residues occur in four regions, each of which contains three or four tryptophan residues separated by 0-12 other residues. This clustering is significant because both histidine and tryptophan have been implicated in the binding of heme. A computer analysis did not identify significant matches of human hemopexin to any protein, including cytochromes and other heme-binding proteins, which suggests that the human hemopexin gene evolved from a unique primordial gene differing from those of other heme-binding proteins.     

Hypothesis about the function of membrane-buried proline residues in transport proteins.

In a survey of the bilayer-spanning regions of integral membrane proteins, membrane-buried proline residues were found in nearly all transport proteins examined, whereas membrane-buried regions of nontransport proteins were largely devoid of intramembranous proline residues. When amino acids from the complete sequences of representative sets of transport and nontransport membrane proteins were analyzed for the distribution of proline residues between aqueous vs. membranous domains, proline was shown to be selectively excluded from membranous domains of the nontransport proteins, in accord with expectation from energetic and structural considerations. In contrast, proline residues in transport proteins were evenly distributed between aqueous and membranous domains, consistent with the notion that functional membrane-buried proline residues are selectively included in transport proteins. As cis peptide bonds involving proline arise in proteins and have been implicated in protein dynamic processes, the cis-trans isomerization of an Xaa-Pro peptide bond (Xaa = unspecified amino acid) buried within the membrane--and the resulting redirection of the protein chain--is proposed to provide the reversible conformational change requisite for the regulation (opening/closing) of a transport channel. Parallel to this function, the relatively negative character of the carbonyl groups of Xaa-Pro peptide bonds may promote their participation as intramembranous liganding sites for positive species in proton/cation transport processes.     

Methionine residues as endogenous antioxidants in proteins

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they can form disulfide bonds that contribute to protein structure. In contrast, the specific functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, available as an efficient oxidant scavenger. Reduction back to methionine by methionine sulfoxide reductases would allow the antioxidant system to function catalytically. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the 16 methionine residues could be oxidized with little effect on catalytic activity of the enzyme. The oxidizable methionine residues were found to be relatively surface exposed, whereas the intact residues were generally buried within the core of the protein. Furthermore, the susceptible residues were physically arranged in an array that guarded the entrance to the active site.     

Involvement of tyrosine residues in the tanning of proteins by 3-hydroxyanthranilic acid

The binding of oxidized phenolic compounds to proteins is of importance in a number of biological systems, including the sclerotization of insect cuticle and the tanning of cocoons. 3-Hydroxyanthranilic acid (3HAA), an aminophenol, is a tryptophan metabolite that undergoes autoxidation readily, and proteins incubated in the presence of 3HAA and oxygen become colored and oxidized. Some moth species are thought to employ this reactivity of 3HAA with proteins for the tanning of cocoons, but the detailed mechanism of this process has not been studied previously. We show that one reaction pathway involves the covalent coupling of 3HAA with tyrosine to form a benzocoumarin derivative, a dibenzo[b,d]pyran-6-one. The stability of the benzocoumarin to conditions of acid hydrolysis normally used for protein digestion has enabled the isolation of the tyrosine adduct from bovine serum albumin that had been incubated with 3HAA. The adduct was also isolated from cocoons of Samia cynthia and Hyalophora gloveri, two species of moths reported to utilize 3HAA for cocoon tanning. These findings indicate that one mechanism of interaction of 3HAA with proteins involves a radical-radical coupling with tyrosine residues.     

Multiple-basin energy landscapes for large-amplitude conformational motions of proteins: Structure-based molecular dynamics simulations

Biomolecules often undergo large-amplitude motions when they bind or release other molecules. Unlike macroscopic machines, these biomolecular machines can partially disassemble (unfold) and then reassemble (fold) during such transitions. Here we put forward a minimal structure-based model, the "multiple-basin model," that can directly be used for molecular dynamics simulation of even very large biomolecular systems so long as the endpoints of the conformational change are known. We investigate the model by simulating large-scale motions of four proteins: glutamine-binding protein, S100A6, dihydrofolate reductase, and HIV-1 protease. The mechanisms of conformational transition depend on the protein basin topologies and change with temperature near the folding transition. The conformational transition rate varies linearly with driving force over a fairly large range. This linearity appears to be a consequence of partial unfolding during the conformational transition.     

Disturbances of tryptophan metabolism in mice acutely deprived of tryptophan

The effect of a dietary tryptophan deficiency on tissue serotonin (5-hydroxytryptamine) synthesis rates, systemic tryptophan metabolism and its response to steroid or cycloheximide treatment was investigated. Brain serotonin synthesis was depressed in tryptophan-deprived (TD) mice while duodenal serotonin synthesis was enhanced following a tryptophan load. Liver total protein content was initially depressed in TD mice but increased following a tryptophan load. Blood tryptophan and albumin were depressed in TD mice while the percentage of albumin-bound tryptophan significantly increased in TD mice. Serum-free fatty acids were not significantly altered. Furthermore, enzyme kinetics studies indicated that in TD mice, tryptophan-5-hydroxylase has a reduced Vmax, while the Km for both tryptophan and the pteridine cofactor was significantly lowered. The tryptophan hydroxylase response to tryptophan or hydrocortisone injection was accentuated in TD mice while the tryptophan-2,3-dioxygenase response to tryptophan or hydrocortisone injection was blunted in TD mice. Finally, injection of tryptophan and cycloheximide blocked the tryptophan-2,3-dioxygenase response while the tryptophan hydroxylase response was unaltered in both control and TD diet mice.     

Relevance of free tryptophan in serum to tissue tryptophan concentrations

The consumption of a carbohydrate diet by fasted rats is followed by major decreases in serum nonesterified fatty acids (NEFA) and nonalbumin-bound tryptophan (unbound tryptophan), but by increases in serum total tryptophan and brain tryptophan; the tryptophan concentrations of liver and small intestine are unchanged, while that of skeletal muscle falls slightly. The addition of 15% or 30% fat to a protein-carbohydrate diet results in dose-related increases in serum NEFA and serum unbound tryptophan, but no significant changes in serum total tryptophan or brain tryptophan. The observation that diet-induced changes in serum unbound tryptophan does not correlate with brain tryptophan concentrations is independent of the method used to separate free from albumin-bound serum tryptophan. These studies confirm that, in the rat, a major physiologic regulator of the extent to which serum tryptophan binds to albumin is the concentration of NEFA in serum. These studies also provide additional evidence that the concentration of tryptophan in the brain is not necessarily determined by the size of the unbound pool of tryptophan in blood as measured in serum.     

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.Proteins exert the majority of their functions in the form of protein complexes to control cellular signaling, protein synthesis, folding and degradation, and many more essential processes. Therefore, elucidating the composition and structure of such complexes has been a longstanding goal of biological research.MS-based proteomics has emerged as one of the main techniques to identify and quantify proteins and their modifications in biological samples such as isolated complexes, proteome fractions, or whole proteomes. Various MS methods now provide structural information on protein assemblies (1–3). Among them, chemical cross-linking and identification of cross-linked peptides by MS (XL-MS) has been increasingly applied to determine the subunit arrangements of biologically relevant complexes (4–6). Such XL-MS experiments indicate the locations of cross-linking sites and thus the spatial proximity of reactive groups that are connected by a covalent bond. This information is then used to determine the positioning of subunits or locate interacting regions, alone or in combination with other techniques such as NMR spectroscopy, electron microscopy, and X-ray crystallography.In the last few years, optimized protocols and new computational tools for the reliable analysis of XL-MS datasets resulted in significant advances of the XL-MS technology (4–6). These advances have contributed to the emergence of a robust, integrated XL-MS method that has been successfully applied for structure determination of a number of large protein complexes (7–11) and the detection of direct, physical interactions in whole cells (12–14). To date, the cross-linking chemistries applied in these studies have targeted primary amines. Predominantly, N-hydroxysuccinimide esters were used as reactive groups, although other chemistries, for example, based on amidates, have also been described (8, 15, 16). Cysteine-specific cross-linking is also well established but usually does not yield sufficient structural information due to the low prevalence of cysteines in proteins and their involvement in the formation of disulfide bonds. Zero-length cross-linking by carbodiimide coupling (17–19) and photochemical cross-linking (20) are other strategies that have been described but have not yet found widespread application in the field.The development of cross-linking chemistries that cross-link functional groups different from amino groups but maintain the efficiency achieved by amine-specific cross-linking are expected to be highly beneficial to increase the depth of structural information obtained from cross-linking experiments. Specifically, such a technique would generate distance restraints from protein regions that are refractive to amine-specific cross-linking under the conditions used and reduce the coverage bias in basic sites. Also, typically only a fraction of theoretically possible cross-links are experimentally observed. This effect is presumably caused by variations in the reactivity of individual lysine residues and/or the unsuitable properties of the resulting cross-linked peptides for MS analysis. An increase in the number of cross-links per substrate by the use of an orthogonal chemistry would therefore be beneficial for de novo identification of hetero-oligomer subunit architectures, as well as restraints in hybrid methods incorporating low-resolution structural information.Residues with carboxyl-terminating side-chains (aspartic and glutamic acids) are attractive targets because of their high prevalence in most proteins. In the most recent release of the SwissProt database (21), version 2014_04, 5.5% and 6.7% of all residues are Asp and Glu, respectively, compared with 5.8% for Lys. However, the low intrinsic chemical reactivity of carboxylic acids poses practical challenges for cross-linking reactions, requiring the use of a coupling reagent. Novak and Kruppa used different dihydrazides as cross-linking reagents in combination with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for activation (22). However, to obtain sufficiently high reaction yields, cross-linking was carried out at relatively low pH (5.5 in ref. 22), which is incompatible with pH-sensitive assemblies. Furthermore, the method was only applied to a single protein, ubiquitin, resulting in the identification of two cross-links.Here we introduce a cross-linking chemistry that connects proximal carboxyl groups [acidic cross-linking (AXL)], whereby side-chains of Asp and Glu residues are cross-linked with dihydrazides using the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (23) (Fig. 1A). In contrast to EDC, DMTMM is able to couple carboxylic acids with hydrazide-based cross-linkers at neutral pH (7–7.5), ensuring good reaction yields and biocompatibility. Experiments with model proteins yielded numbers of cross-linked peptides that were in the same range to those generated by the well-established Lys-specific cross-linking using the reagent disuccinimidyl suberate (DSS). On top of that, a second set of cross-linking restraints is observed in the form of zero-length cross-links between Lys and Asp or Glu residues, respectively.Open in a separate windowFig. 1.(A) Cross-linking reactions involving coupling of carboxyl groups using dihydrazides (acidic cross-linking, AXL; Top) and zero-length cross-linking (ZLXL) with DMTMM as coupling reagent. R¹ and R² denote acidic residues (Asp, Glu) in a single or two different proteins, R³ denotes a primary amine (usually Lys). (B) Structure of the two dihydrazide reagents used in this study. Asterisks denote positions where hydrogen atoms are exchanged for deuterium atoms in the heavy form of the reagent. The spacer length of the reagents (calculated between the terminal nitrogen atoms) is also given.To show the practical relevance of the method, we applied it to multisubunit complexes in the megadalton range that have been recently probed with lysine-specific cross-linking and for which structural information is available. The results indicate that for both the chaperonin TRiC/CCT from Bos taurus and the Schizosaccharomyces pombe 26S proteasome, cross-links were identified that are in agreement with the available structures of these complexes. Acidic and zero-length cross-links provided orthogonal sets of structural restraints that are complementary to a cross-linking chemistry targeting lysines. We therefore expect that chemical cross-linking of acidic residues will become an important method for a more comprehensive structural analysis of protein complexes by XL-MS.     

Increased oxidized methionine residues in BAL fluid proteins in acute or chronic bronchitis.

Phagocytic cells such as alveolar macrophages (AM) or polymorphonuclear neutrophils (PMN) in the bronchoalveolar tract are a potential source of the oxygen-derived free radicals which are presumed to be involved in lung tissue damage. Previous results have shown that the methionine sulphoxide (MET(O)) content of bronchoalveolar lavage fluid (BALF) protein is a reliable parameter to indicate oxidative processes in idiopathic pulmonary fibrosis (IPF). We measured the molar ratio between MET(O) and methionine (MET) in the BALF protein from healthy nonsmokers (control group), healthy smokers and patients with acute or chronic bronchitis (AB or CB). The MET(O)/MET ratio of the nonsmoking group (n = 11) was 0.046 +/- 0.008 (mean +/- SEM). Healthy smokers (n = 8) had similar values (0.042 +/- 0.008), even though they had strongly increased AM counts in BALF. Patients with AB (n = 12) showed an increased MET(O)/MET ratio (0.191 +/- 0.031) and had high PMN but normal AM counts in BALF. Patients with CB (n = 13) showed an increase in the MET(O)/MET ratio (0.086 +/- 0.010) and moderately increased PMN and markedly increased AM counts. Taking all results together, the MET(O)/MET ratio correlated positively with the relative PMN number (r = 0.70; p less than 0.0002) and inversely with the relative AM number (r = 0.67; p less than 0.0002). In the group with CB, the MET(O)/MET ratio correlated inversely with forced expiratory volume in one second (FEV1) % pred. (r = -0.77) and FEV1/inspiratory vital capacity (IVC) % pred. (r = -0.89).(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlated dynamics of consecutive residues reveal transient and cooperative unfolding of secondary structure in proteins

Nuclear spin relaxation is a powerful method for studying molecular dynamics at atomic resolution. Recent methods development in biomolecular NMR spectroscopy has enabled detailed investigations of molecular dynamics that are critical for biological function, with prominent examples addressing allostery, enzyme catalysis, and protein folding. Dynamic processes with similar correlation times are often detected in multiple locations of the molecule, raising the question of whether the underlying motions are correlated (corresponding to concerted fluctuations involving many atoms distributed across extended regions of the molecule) or uncorrelated (corresponding to independent fluctuations involving few atoms in localized regions). Here, we have used (13)C(alpha)(i - 1)/(13)C(alpha)(i) differential multiple-quantum spin relaxation to provide direct evidence for correlated dynamics of consecutive amino acid residues in the protein sequence. By monitoring overlapping pairs of residues (i - 1 and i, i and i + 1, etc.), we identified correlated motions that extend through continuous segments of the sequence. We detected significant correlated conformational transitions in the native state of the E140Q mutant of the calmodulin C-terminal domain. Previous work has shown that this domain exchanges between two major conformational states that resemble the functionally relevant open and closed states of the WT protein, with a mean correlation time of approximately 20 micros. The present results reveal that an entire alpha-helix undergoes partial unraveling in a transient and cooperative manner.     

Positively charged amino acid residues can act as topogenic determinants in membrane proteins.

When alkaline phosphatase is fused to the periplasmic domain of a cytoplasmic membrane protein, it is efficiently exported to the periplasm. Such a hybrid protein exhibits high alkaline phosphatase enzymatic activity. When alkaline phosphatase is fused to the cytoplasmic domain of a membrane protein, it remains, for the most part, in the cytoplasm. Such fusions exhibit low enzymatic activity. However, stable retention of alkaline phosphatase in the cytoplasm requires the presence in the fusion protein of the cytoplasmic loop ordinarily present in that position in the native, unfused protein. Using oligonucleotide-directed mutagenesis, we have shown that positively charged amino acids are required for the stable cytoplasmic localization of the fused alkaline phosphatase. We propose that, in addition to hydrophobic transmembrane segments, positively charged amino acids in the hydrophilic cytoplasmic domains of a membrane protein are determinants of the protein's topology.