Protein Higher Order Structure Research Articles

Direct Enzymatic Digestion of Protein Complexes for MS Analysis

INTRODUCTIONThe individual components of a purified protein complex can be determined by MS. Components can be separated by SDS-PAGE and subsequently digested, or the entire protein complex can be digested by chemical agents or by proteases. The mixture of peptides that results from digestion without prior separation is more complex than that obtained from the digestion of a single protein, and can best be resolved subsequently using HPLC in combination with a biphasic column. Protein complexes can be digested directly using several different protocols. Common methods include a double digest with endoproteinase Lys-C and trypsin, or a triple digest with elastase, subtilisin, and trypsin. Endoproteinase Lys-C cleaves carboxy-terminal to lysine residues, while trypsin cleaves carboxy-terminal to lysine and arginine residues. The triple digest uses the relatively nonspecific proteases elastase and subtilisin to create a large number of overlapping peptides for the analysis of post-translational modifications. The double and triple digest protocols begin with the reduction and alkylation of cysteine residues to disrupt disulfide bonds and, therefore, higher-order protein structure.

Solution and gas phase hydrogen/deuterium exchange of oligoamide complexes by nanoelectrospray mass spectrometry

Hydrogen bonding is crucial to biological systems and is intrinsic to non-covalent interactions. They are important in the formation of higher order structures of proteins as well as in the interactions of proteins with other biological molecules. Electrospray ionization (ESI) is an important tool in the study and characterization of molecular complexes and has proven to be extremely powerful and invaluable in the studying of biomolecular structures and non-covalent interactions. We have utilized solution and gas phase hydrogen/deuterium exchange (HDX) as a method to determine the specificity of supramolecular complex formation using monomers possessing sites containing hydrogen-bond donor and acceptor groups. By comparing the average number of exchanges for the monomer subunits to the average number of exchanges for the complex, we can distinguish if a specific complex is formed in solution, or whether it is the artifact of a gas phase process during ESI. In this paper we have investigated several non-covalent supramolecular complexes by nanoelectrospray (nanoESI) mass spectrometry (MS). By using the solution and gas phase HDX, we were able to identify several specific supramolecular complexes. Thus, solution and gas phase HDX combined with nanoESI-MS provides for a convenient method in ascertaining the origin and stability of non-covalent complexes.

Stabilization of bzip Peptides through Incorporation of Fluorinated Aliphatic Residues

Two fluorinated amino acids, 5,5,5-trifluoroisoleucine (5TFI) and (2S,3R)-4,4,4-trifluorovaline (4TFV), which have been shown to serve as isoleucine surrogates in protein synthesis in Escherichia coli, have been incorporated in vivo into basic leucine zipper (bzip) peptides derived from GCN4. The extents of residue-specific incorporation of 5TFI and 4TFV were 90 and 88 %, respectively, of the encoded isoleucine residues, as evidenced by MALDI mass spectrometry and amino acid analysis. Both circular dichroism and equilibrium sedimentation studies of the fluorinated bzip peptides indicated preservation of secondary and higher-order protein structure. Thermal-denaturation experiments showed an increase of 27 degrees C in melting temperature when isoleucine was replaced by 5TFI. However, the T(m) of the peptide containing 4TFV was increased by only 4 degrees C over that of the peptide containing valine. Similar trends were observed in chemical denaturation studies in which DeltaDeltaG(unfold) in water was determined to be 2.1 or 0.3 kcal mol(-1) upon incorporation of 5TFI or 4TFV, respectively. When the fluorinated peptides were tested for DNA binding, both their affinity and specificity were similar to those of the respective hydrogenated peptides. These results suggest that fluorinated amino acids, even when introduced into the same positions, can have markedly different effects on the physical properties of proteins, while having little impact on secondary and higher-order structure.

Hollow‐Fiber Flow Field‐Flow Fractionation: A Gentle Separation Method for Mass Spectrometry of Native Proteins

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.

Characterization of hypervariable region in hepatitis C virus envelope protein during acute and chronic infection

Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure. In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.

D-Amino Acid in Elderly Tissues

All living organisms on earth are composed of L-amino acids and D-sugars. Therefore the presence and function of D-amino acids in living organisms have not been studied. Recently, however, D-aspartic acid (D-Asp) which is a D-amino acid, has been detected in various proteins from human tissue samples such as eye lens, brain, skin, bone, teeth, aorta from elderly individuals. It has been explained that the presence of D-Asp is the result of racemization of Asp residues in the protein during the life span, inasmuch as the proteins in these tissues are metabolically inert. The Asp-151 and Asp-58 residues in alpha A-crystallin from elderly human lens are especially stereochemically labile and the D/L ratios of these residues were greater than 1.0. A D/L ratio greater than 1.0 is not defined as racemization, but as the inversion of configuration. This was the first observation that inversion occurred in the configuration of amino acids in vivo during the natural aging process. In this review, we summarize the D-Asp in the various tissues reported by many researchers and describe the mechanism of D-Asp formation in protein. We suggest that a chiral reaction field exists in the native higher order structure of protein which induces the inversion of L-Asp to D-Asp residue.

Higher order structure of proteins solubilized in AOT reverse micelles

The higher order structure of proteins solubilized in an bis(2-ethylhexyl) sulfosuccinate sodium (AOT) reverse micellar system was investigated. From circular dichroic (CD) measurement, CD spectra of cytochrome c, which is solubilized at the interface of reverse micelles, markedly changed on going from buffer solution to the reverse micellar solution, and the ellipticity values in the far- and near-UV regions decreased with decreasing the water content ( W 0: molar ratio of water to AOT), indicating that the secondary and tertiary structures of cytochrome c changed with the water content. The ellipticity of ribonuclease A, which is solubilized in the center of micellar water pool, in the near-UV region was dependent on W 0 and became minimum when W 0 of ca. 8 while the ellipticity in the far-UV region was almost constant, indicating that the tertiary structure of ribonuclease A was affected by the water content, but the secondary structure was conserved. The degree of curvature of the micellar interface appears to influence the protein structure because the reverse micelle size is linearly proportional to the W 0 value. As evidence of this, when the micelle size was comparable to the protein's dimensions, the structures were more affected by the water content. Judging from the dependence of the factor influencing the protein structure on the protein species, the location of solubilized protein in reverse micelles is significantly related to whether the protein structure in the system is affected by the micellar interface. In the cases of cytochrome c and lysozyme, the ellipticity against W 0 was dependent on the AOT concentration. In contrast, ribonuclease A gave very similar ellipticity values whatever the AOT concentration. In the n-hexane micellar system, cytochrome c exhibited lower ellipticity values and ribonuclease A in the lower W 0 range ( W 0 < ca. 8) higher ellipticity values. These results indicated that the interaction between the protein and the micellar interface is a dominant factor influencing the protein structure in reverse micelles, and that it is governed by the location of solubilized proteins and the state of the micellar interface.

Protein structure characterization with mass spectrometry

Mass spectrometry is now commonly being used to determine both the primary and higher order structures of proteins. The basis for these investigations lies in the ability of mass analysis techniques to detect changes in protein conformation under differing conditions. These experiments can be conducted on proteins alone (with no modifying substance present) or in combination with proteolytic digestion or chemical modification. In addition to primary structure determination, proteases and chemical modification have long been used as probes of higher order structure, an approach that has been recently rejuvenated with the emergence of highly sensitive and accurate mass analysis techniques. Here, we review the application of proteases as probes of native structure and illustrate key concepts in the combined use of proteolysis, chemical modification, and mass spectrometry. For example, protein mass maps have been used to probe the structure of a protein/protein complex in solution (cell cycle regulatory proteins, p21 and Cdk2). This approach was also used to study the protein/protein complexes that comprise viral capsids, including those of the common cold virus where, in addition to structural information, protein mass mapping revealed mobile features of the viral proteins. Protein mass mapping clearly has broad utility in protein identification and profiling, yet its accuracy and sensitivity is also allowing for further exploration of protein structure and even structural dynamics.

Correlated Mutation Analysis of C2H2 Zinc Finger Domains

Zinc finger proteins perform various functions in cellular processes, such as transcription, translation, metabolism and signaling, by binding to nucleic acids or proteins. In a zinc finger domain, a zinc ion forms four coordinate bonds for its stable fold. A C2H2 domain has approximately 25 to 30 amino acid residues, including paired cysteines and histidines as zinc coordinating residues and possessing two short β-strands followed by an α-helix. Most C2H2 zinc finger proteins bind to DNA. Many of the proteins also bind to RNA or proteins. The binding properties depend on the amino acid residues of the zinc finger loop, the arrangement of C2H2 domains in proteins and the higher order structure of proteins [2]. In this research, we analyze C2H2 domains using a method of correlated mutation analysis(CMA). There has been several CMA methods based on substitution matrices [1] or likelihood matrices derived from previously solved structures [3]. We present a CMA method using a mixture of canonical distributions.

Direct detection of the protein quaternary structure and denatured entity by small-angle scattering: guanidine hydrochloride denaturation of chaperonin protein GroEL

A change in the higher-order structure of an oligomeric protein is directly detectable by small-angle scattering. A small-angle X-ray scattering (SAXS) study of the denaturation process of the chaperonin protein GroEL by guanidine hydrochloride (GdnHCl) showed that the disappearance of the quaternary structure can be monitored by using a Kratky plot of the scattered intensities, demonstrating the advantage of the SAXS method over other indirect methods, such as light scattering, circular dichroism (CD), fluorescence and sedimentation. The collapse of the quaternary structure was detected at a GdnHCl concentration of 0.8 Mfor a solution containing ADP (adenosine diphosphate)/Mg2+(2 mM)/K+. From pairwise plots of the change in forward scattering intensityJ(0)/C(weight-average molecular weight) and thez-average (root mean square) radius of gyration against the GdnHCl concentration, the stability and nature of the denatured protein can be determined. The SAXS results suggest that the GroEL tetradecamer directly dissociates to the unfolded coil without going through a globular monomer state. The denatured ensemble is not a single unfolded monomer coil particle, but some mixture of entangled aggregates and a monomer of the coil molecules. Small-angle scattering is a powerful method for the detection and study of changes in quaternary and higher-order structures of oligomeric proteins.

Directed molecular evolution of ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase catalyzes a rate-limiting reaction in prokaryotic glycogen and plant starch biosynthesis. Despite sharing similar molecular size and catalytic and allosteric regulatory properties, the prokaryotic and higher plant enzymes differ in higher-order protein structure. The bacterial enzyme is encoded by a single gene whose product of ca. 50,000 Da assembles into a homotetrameric structure. Although the higher plant enzyme has a similar molecular size, it is made up of a pair of large subunits and a pair of small subunits, encoded by different genes. To identify the basis for the evolution of AGPase function and quaternary structure, a potato small subunit homotetrameric mutant, TG-15, was subjected to iterations of DNA shuffling and screened for enzyme variants with up-regulated catalytic and/or regulatory properties. A glycogen selection/screening regimen of buoyant density gradient centrifugation and iodine vapor colony staining on glucose-containing media was used to increase the stringency of selection. This approach led to the isolation of a population of AGPase small subunit homotetramer enzymes with enhanced affinity toward ATP and increased sensitivity to activator and/or greater resistance to inhibition than TG-15. Several enzymes displayed a shift in effector preference from 3-phosphoglycerate to fructose-6 phosphate or fructose-1,6-bis-phosphate, effectors used by specific bacterial AGPases. Our results suggest that evolution of AGPase, with regard to quaternary structure, allosteric effector selectivity, and effector sensitivity, can occur through the introduction of a few point mutations alone with low-level recombination hastening the process.

Investigating the higher order structure of proteins. Hydrogen exchange, proteolytic fragmentation, and mass spectrometry.

It has been apparent for some years that the structures of proteins are dynamic rather than static. For some proteins, dynamics is essential to function (e.g., refs. 1–7). These structural changes have been detected for more than 30 yr by observing hydrogen exchange between peptide amide hydrogens and solvent containing the hydrogen isotopes tritium or deuterium (8–10). Although tritium is no longer used extensively for this purpose, deuterium is widely used in hydrogen exchange studies, especially in multidimensional nuclear magnetic resonance (NMR), in which amide hydrogen signals disappear on deuteration. Since deuterium weighs 1 Dalton more than protium, hydrogen exchange in proteins can also be detected by mass spectrometry. This approach is complementary to NMR in some respects and clearly advantageous in others.

Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.

Multiple sequence alignment: Algorithms and applications

Elucidation of interrelationships among sequence, structure, function, and evolution (FESS relationships) of a family of genes or gene products is a central theme of modern molecular biology. Multiple sequence alignment has been proven to be a powerful tool for many fields of studies such as phylogenetic reconstruction, illumination of functionally important regions, and prediction of higher order structures of proteins and RNAs. However, it is far too trivial to automatically construct a multiple alignment from a set of related sequences. A variety of methods for solving this computationally difficult problem are reviewed. Several important applications of multiple alignment for elucidation of the FESS relationships are also discussed. For a long period, progressive methods have been the only practical means to solve a multiple alignment problem of appreciable size. This situation is now changing with the development of new techniques including several classes of iterative methods. Today's progress in multiple sequence alignment methods has been made by the multidisciplinary endeavors of mathematicians, computer scientists, and biologists in various fields including biophysicists in particular. The ideas are also originated from various backgrounds, pure algorithmics, statistics, thermodynamics, and others. The outcomes are now enjoyed by researchers in many fields of biological sciences. In the near future, generalized multiple alignment may play a central role in studies of FESS relationships. The organized mixture of knowledge from multiple fields will ferment to develop fruitful results which would be hard to obtain within each area. I hope this review provides a useful information resource for future development of theory and practice in this rapidly expanding area of bioinformatics.

Multiple sequence alignment as a crossroad of biological macromolecular information.

Elucidation of interrelationship among sequence, structure, function, and evolution of a family of genes or gene products is a central theme of modern molecular biology. Multiple sequence alignment is a powerful tool for such many-sided studies. We have developed a new strategy called "doubly nested randomized iterative method" to tackle this computationally hard problem. When tested upon structural alignments as references, our method was proven to perform significantly better than currently most popular methods. Our method will be applicable to a variety of problems, such as phylogenetic reconstruction, and prediction of functionally important regions and higher order structures of proteins.

Molecular chaperones and the cytoskeleton

Heat shock proteins, first observed because they are preferentially synthesized by organisms exposed to heat or other physiological stress, are also synthesized constitutively. These proteins are divided into several families, namely, HSP100, 90, 70, 60 (chaperonin), and the small heat shock/alpha-crystallin proteins. They enjoy a wide phylogenetic distribution and are important because they function as molecular chaperones, able to mediate many cellular processes through an influence on higher order protein structure. For example, molecular chaperones assist in the transport of proteins into mitochondria and chloroplasts, as well as influencing clathrin lattice dynamics, viral replication and transcriptional activation. Under conditions of stress, some molecular chaperones prevent denaturation of proteins while others may dissociate protein aggregates, refolding monomers derived therefrom or directing their proteolytic destruction. We present in this review an analysis of the emerging literature on the relationship between molecular chaperones and the cytoskeleton, a collection of polymeric structures consisting of microtubules, microfilaments and intermediate filaments. A recent development in this field is identification of the TCP-1 complex as the eukaryotic cytoplasmic chaperonin which directs folding of cytoskeletal proteins such as alpha/beta/gamma-tubulin, actin and centractin. Moreover, the TCP-1 complex is a centrosomal component, apparently involved in the nucleation of microtubules. Other molecular chaperones recognize one or more cytoskeletal elements and in most cases they modulate the assembly of and/or provide protection for their constituent proteins. For example, HSP70 protects the centrosome and perhaps intermediate filaments during heat shock, and like HSP90, it binds to microtubules. Small heat shock proteins interact with microfilaments and intermediate filaments, affect their polymerization and guard them from heat shock by a phosphorylation-dependent mechanism. We conclude that molecular chaperones have different but cooperative roles in the formation and function of the eukaryotic cell cytoskeleton.

Mass spectrometry as a readout of protein structure and function.

Proteins have evolved to carry out very specific functions within the cell by interacting with a diverse set of biomolecules. Understanding how a protein's higher order structure relates to its function is important for defining the molecular basis of these interactions. In recent years, mass spectrometry has become an important tool for dissecting protein structure and function. Using electrospray ionization (ESI)- and matrix-assisted laser desorption/ionization (MALDI)-based approaches, it has been possible to monitor protein folding, characterize noncovalent protein complexes, and assess the contribution of individual amino acid residues to a protein's function. Here, it is our goal to summarize these approaches and highlight recent, biologically relevant applications where mass spectrometry has provided unique insight into the mysteries of protein structure and function.

The use of bioreactive probes in protein characterization.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has in the past decade found routine use in the biological sciences. With this use has evolved several mass spectrometric-based methods directed at the intricate investigation of biomolecular structure and function. One such methodology involves the enzymatic modification of a protein prior to the mass spectrometric readout of the resulting products. The enzyme-modification/mass spectrometric approach has a definite use in a number of applications, including: the verification/identification of protein sequence, elucidation of post-translational modifications, the investigation of protein higher-order structure, and even the characterization of the modifying enzyme. To avoid the potentials of sample loss and autolytic interferences in the mass spectrum, mass spectrometer targets can be covalently derivatized with enzymes for use in the characterization procedures. The enzymatically active, or bioreactive, probes are used by application of the analyte to the activated surface, followed by application of a suitable MALDI matrix and mass analysis from the surface of the probe. Limited transfer and handling steps eliminate sample losses, and surface-tethered enzymes (and autolytic fragments) are prohibited from interfering with analytical signals in the mass spectra. In addition, the probes are rapid and easy to use. Reviewed here are issues of concern during the manufacture and use of the bioreactive probes, and application of the probes to investigate protein structure and function.

HSP100/Clp proteins: a common mechanism explains diverse functions

The HSP100/Clp proteins are a newly discovered family with a great diversity of functions, such as increased tolerance to high temperatures, promotion of proteolysis of specific cellular substrates and regulation of transcription. HSP100/Clp proteins are also synthesized in a variety of specific patterns and, in eukaryotes, are localized to different subcellular compartments. Recent data suggest that a common ability to disassemble higherorder protein structures and aggregates unifies the molecular functions of this diverse family.

Synthetic molecules that fold into a pleated secondary structure in solution

THE construction of synthetic molecules that fold or assemble predictably into large, well defined structures represents a fertile area of chemistry. Many supramolecular systems have been reported that self-assemble as a result of non-covalent interactions1-7; and the control of higher-order protein structure by de novo design has also been demonstrated8,9. Protein secondary structural motifs have also been stabilized by incorporating artificial groups that impose constraints on the folded architecture10-12. Here we describe the synthesis of molecules that will fold in water into a pleated structure, as a result of interactions between alternating electron-rich donor groups and electron-deficient acceptor groups. We verify the pleated structure using absorption and NMR spec-troscopy. Donor-acceptor interactions have been used previously to engineer specific supramolecular geometries2,13, and are energetically favourable in organic as well as in aqueous solutions. But whereas previously such interactions have been used to effect self-assembly of distinct molecules, our results show that they can also determine the secondary structure of complex synthetic molecules in solution.