Molecular Imaging: FRET Microscopy and Spectroscopy

Texas

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Proteins and the Flow of Information in Cellular Function

ROBERT H. KRETSINGER

1. INTRODUCTION

Förster resonance energy transfer (FRET) microscopy provides many beautiful and informative images, most of them focusing on protein complexes. What is the appropriate framework to understand these data? This introductory chapter argues that the flow of information provides that concept. Further, the synergism of the perspectives of molecular structure and of emergent properties of complex systems is essential to understanding this flow. This chapter should provide basic ideas and vocabulary to microscopists who are not so familiar with the concepts or jargon of protein structure, function, and evolution and of information flow.

2. PROTEINS AND INFORMATION

Francis Crick (1958) posited in his Central Dogma that DNA makes RNA makes protein. This has subsequently been elaborated to include reproduction, DNA makes DNA, and in some viruses, RNA makes RNA. The discovery of reverse transcriptase by Baltimore (1970) and by Temin and Mizutani (1970) necessitated adding RNA makes DNA (Fig. 1-1). However, the fundamental assertion that genetic information flows, makes in the vernacular quoted above, from nucleic acid to protein and not in the reverse sense remains one of the fundamental tenets of molecular biology and has driven much of its research over the past half century.

FIGURE 1-1 Flow of information. The scheme originally suggested by Crick (1958), that DNA makes RNA makes protein. The essential point is that genetic information encoded in the sequence of nucleic acids can be transduced to the sequence of amino acids in proteins but not in the reverse direction. A additional transmission of discrete sequence information: reverse transcription of RNA to DNA and replication, DNA makes DNA and RNA makes RNA, in solid lines. Proteins in turn can act on and change in a continuous manner the information content of itself or of other proteins, RNA, and DNA, either by covalent modification or simply binding reversibly, all indicated by dashed lines. Further, proteins can change the content and flow of information in a cellular mileau by catalysis, generating metabolites, altering ion fluxes, or assembling structures as membranes and cell walls, all indicated by dotted lines.

The strong corollary of the Central Dogma is that proteins represent the molecular phenotype of the cell and of the organism. Two exceptions to this approximation are especially fascinating. Ribosomal RNAs (rRNA) and transfer RNAs (tRNA) play essential roles in protein synthesis and probably reflect the dominant role of catalytic RNAs in ancient cells. In addition to other catalytic functions, for example, of spliceosomes (Rappsilber et al., 2002), there are several recently discovered classes of small, interfering RNAs (siRNA) and of microRNAs (miRNA) that can modulate the functions of messenger RNAs (mRNA).

Important as these non-messenger RNAs are, the generalization that proteins represent the cell’s phenotype remains valid. Certainly, there are components of cells and extracellular materials other than proteins—lipids, carbohydrates, metabolites, and minerals—in addition to proteins that have undergone various posttranslational modifications (Nakai, 2001). Nonetheless, these components and modifications ultimately reflect the actions of proteins, hence the emphasis over the past fifty years on identifying and characterizing proteins.

3. PROPERTIES OF PROTEINS, THE REDUCTIONIST APPROACH

Scientists have long studied proteins in terms of their amino acid sequences, enzymatic activities, thermodynamic properties, and atomic structures. This has yielded a trove of empirical data from which general principles have emerged. Yet, increasingly one comes to appreciate that the character and function of a cell cannot now or in the foreseeable future be deduced or predicted from the properties of its constituent proteins. The cell’s phenotype is an emergent property, certainly consistent with and explained in terms of these characteristics, but not predictable from them. It is essential that one understand these basic properties, a few of which are summarized in this section. However, one must also measure the indirect and direct interactions of these proteins over time—that is, the further flow of information in the cell beyond translation. This is the subject of the next section and of the other chapters in this book.

The (almost) complete genomic sequences of 18 archae, 141 bacteria, and 22 eukaryotes (http://www.ebi.ac.uk/genomes/) are available. From these the amino acid sequences of a million proteins, representing over a thousand homolog families, are available. Usually the codons encoding the first (N-terminal) and last (C-terminal) amino acids can be safely inferred, as can intron sites; however, some genes (in this context referring only to those genes encoding proteins) may be missed. Of greater concern, exons are sometimes spliced out of the processed mRNA prior to its translation in the ribosome (Rappsilber, 2002) to a linear sequence of amino acids.

Protein sequences are also deduced from the sequences of mRNA as copied back to complementary DNA (cDNA), using reverse transcriptase in vitro. These sequences should be free of splicing ambiguities and reflect protein that is actually being synthesized in the cell(s) of interest under specified conditions (Scamborova et al., 2004).

Having gotten the amino acid sequences of the expressed proteins, one must appreciate that the linear combinations of 20, genetically encoded amino acids generate a great variety of structures that have a remarkable range of properties. Few of the 20N possible sequences, for a protein N residues long, have ever been synthesized (Di Lallo et al., 2003). Yet several unifying themes, with associated terminology, have emerged.

Anfinsen (1973) demonstrated that RNAase A can be denatured, then renatured in vitro, and will regain full enzymatic activity. This treatment involves no cleavage or formation of covalent bonds, no change in configuration. However, the conformation or rotation about single bonds between carbon, oxygen, nitrogen, and sulfur atoms changes a great deal. One can infer that the conformations of (most) proteins are in their global, free energy minima, and that this conformation is determined solely by the amino acid sequence of the protein in question, even though this has been explicitly demonstrated for only a few proteins.

During the folding process, some cytosolic proteins fall into a local energy minimum in which they are vulnerable to proteolysis. A misfolded protein can diffuse to the interior of a chaperone (Alberti et al., 2002; Phillips et al., 2003), where it is denatured then given another chance to find its energy minimum. If it remains misfolded, it soon has several ubiquitin proteins covalently attached and is thereby labeled for proteolysis in a 26S proteosome (Belz et al., 2002; Woodham et al., 2003).

Proteins synthesized on polyribosomes in the cytosol are released into the cytosol. Those with specific leader sequences bind to receptors on the endoplasmic reticulum and the polypeptide emerging from the ribosome is directed to the lumen of the endoplasmic reticulum. There it is modified, prior to exocytosis, to the extracellular environment or for incorporation as an integral membrane protein. Other leader sequences may target proteins for transport into the matrix or inner membrane of the mitochrondrion, into the nucleus, or to other organelles (Nakai, 2001), such as the parasitophorous vacuole of the malaria parasite, Plasmodium falciparum (Adisa et al., 2003).

Many proteins, regardless of destination within or outside the cell, are subject to post-translational modifications. Examples include (usually) irreversible glycosylation, characteristic of extracytosolic proteins; proteolysis involved in conversion from pre- to pro- to final isoforms often seen in the generation of peptide hormones and neurotransmitters; and reversible phosphorylation of serine, threonine, and tyrosine residues associated with cell signaling. There are scores of such specific modifications, such as ubiquination (mentioned above) and generation of green fluorescent proteins (discussed in later chapters), all of which can significantly affect the physical and functional properties of proteins (Jang and Hanash, 2003).

In addition to the changes in covalent structure, or in configuration, involved in post-translational modification, most proteins reversibly bind ligands (small molecules or ions). This often results in significant changes in conformation and in properties without further change of configuration.

Despite these and many other complications, the inference from Anfinsen’s (1973) experiments remains valid; from the amino acid sequence one should in principle be able to predict the conformation(s) of the protein, excluding modifications and binding of ions or of other molecules. Indeed, if the three-dimensional structure of a homolog is known, one can usually predict the structure assumed by the sequence in question. The closer in sequence these two proteins sharing a common precursor, the more similar in structure (Wood and Pearson, 1999). Correspondingly, the more similar the sequences of the unknown and of the reference, the closer the predicted structure to the real one (Moult et al., 2003; Kretsinger et al., 2004). However, one cannot yet predict with confidence the structure of a new protein ab initio—a very humbling admission—after 30 years of intense effort by the research community. The related question of whether most chains of amino acids randomly assembled by sequence have a distinct structure associated with a distinct energy minimum remains unanswered. Just as one cannot predict ab initio the structure of a protein from its sequence, one also cannot predict the interaction site or affinity of two proteins from their respective structures (Bock and Gough, 2001).

The main chain, or backbone, of a protein can be characterized and computed to excellent approximation by two dihedral angles—ϕ CO, NH-Cα, CO) and ψ(NHi, Cα -CO, Ni+1)—per residue. This is because covalent bond lengths and bond angles are almost constant among amino acids and because the dihedral angle of the peptide bond, ω(Cαn, CO-NH, Cα (n+1)), is planar and (near) constant. The conformations of amino acids as observed in proteins are restricted to small areas of the ϕ, ψ (plot (Ramachandran and Sassiekharan, 1968); this reflects the constraints imposed by van der Waals contacts between atoms of the main chain and between the main-chain and side-chain atoms, primarily Cβ, when ϕ and ψ lie outside of those acceptable ranges (Fig. 1-2). When four consecutive residues have ϕ, ψ values in the H region they form an α helix, characterized by a hydrogen bond from the NH of residue i+4 to the CO of residue i, counting from the N-terminal residue toward the C-terminus. When three consecutive residues have ϕ, ψ s in the S region they form a β strand. Two or more β strands, side by side, either parallel or antiparallel, form a β sheet, which is stabilized by strand-to-strand hydrogen bonds. About a third of residues in globular proteins are involved in α helices, a third in β sheets, and a third in other, imprecisely called turn, or coil, or linker. α Helices tend to be resistant to thermal and chemical denaturation or to proteolysis; proteins with high α -helical content are more stable. Turn regions often provide flexibility between domains, changes in conformation at ligand binding sites, and sites ofproteolysis.

FIGURE 1-2 Ramachandran plot. a: Distribution of ϕ, ψ values observed for all amino acids in proteins determined to high resolution (Hovmöller et al., 2002). Proline is restricted to ϕ ∼ −60° because its side chain forms a five-member pyrrolidine ring involving its α -amine nitrogen. Glycine enjoys a broader range of ϕ, ψ because it has only a hydrogen atom for a side chain. The other 18 amino acids have slighlty different distributions from one another, as if each had a conformational fingerprint. These seven distinct regions in the ϕ, ψ plot account for > 95% of observed conformations of amino acids in crystal structures determined to high resolution; they are assigned identifying symbols: H, residues in α -helices assume this conformation; T, residues in left handed α -helices assume this conformation; T, residues in β-strands assume this conformation; R, residues in polyproline helices assume this conformation; U, a conformation occasionally seen in turns; V, another conformation, distinct from U, occasionally seen in turns; G, a conformation restricted to Gly. b: Conformation of the backbone at ϕ = 180° and ψ = 180°, as found in the β strand. Arrows indicate positive or right-hand, rotation. Rotation about ϕ (NH-Cα) moves Cβ relative to the fixed Cα −1, CO−1, NH, Cα plane. Rotation about ψ (Cα -CO) moves the Cα, CO, NH+1, Cα + 1 plane relative to the fixed Cβ. c: Conformation at ϕ = –109° and ψ = 121°.

A domain of a globular protein, in contrast to a fibrous protein, is often the appropriate unit for analysis from structure to evolution. Most globular proteins consist of several domains, although smaller proteins, such as myoglobin or lysozyme, often consist of a single domain. Most domains under physiological conditions adopt an ensemble of closely related conformations. The axial ratio, from longest to shortest, of a domain is usually less than 3:1. Its constituent chain of residues has a single entrance at its N terminus and a single exit at its C terminus— that is, it could be cut out with two proteolytic clips and assume essentially the same shape as it had when part of the full protein. Often a monomer protein can be cleaved to (or expressed as) its constituent domains then reassembled without formation of covalent bonds between these domains to form a functional protein. This characteristic is exploited in the yeast two-hybrid assay.

Although one speaks of conformation in the singular, nearly all proteins, except a few devoted solely to structure such as collagen or keratin, can assume several closely related conformations. Like nanomachines, these conformations reflect states in the functional cycle of a protein (Valpuesta et al., 2002; Lee et al., 2004). Within a domain the orientations of a few side chains and the conformation(s) of a turn(s) may vary among states. However, the conformations of the domains themselves are usually very similar among these various states of the entire machine. In contrast, the relationships between domains within a multidomain protein may vary significantly because of changes in conformation of the interdomain turns, often called linkers.

The domain is the recognizable unit of evolution. Two domains are homologous if they evolved from a common precursor protein. Since this process was not observed, the burden of proof, or at least of probability, rests with the advocate of homology. Often one asks what the probability is that the domain in question is similar by chance to another domain in a database. If the probability is less than, say, 1 in 10⁵, then the observed similarity is inferred to reflect descent from a common precursor, as opposed to convergent evolution (http://www.ncbi.nlm.nih.gov/BLAST/). Two homologs are referred to as orthologs if they are related by speciation of their host organisms, and as paralogs if they are related by gene duplication. Dendrograms, or phylogenetic trees, of organisms based on their respective orthologs reflect the evolution of those organisms; the more orthologs involved in the analysis, ideally reflecting the entire genome, the more significant the resulting phylogeny (Stuart et al., 2002; Cheng et al., 2003). Dendrograms based on paralogs provide insight into the origins of isoforms, and possible divergence and specialization of function, within that one protein family.

The term protein has several meanings that should be clear from its context. Experimentalists usually encounter the protein replete with post-translational modifications and ligands bound; they deal with the properties thereby imparted. In contrast, geneticists consider the protein according to its sequence of encoded amino acids.

Some proteins function as monomers—a single chain of residues comprised of one or of several domains. Each of these domains in either a homochimeric (homologous domains) or a heterochimeric (different, nonhomologous domains) monomer protein has its own conformation(s) and its own evolutionary history. Most proteins are oligomers. Several identical monomers associate noncovalently to form a homo-oligomer or different monomers form a hetero-oligomer. The monomers of some oligomers associate and dissociate as part of their functional cycles; some associate with much higher affinity. Some monomers, for instance, actin, polymerize to form thin filaments of muscle. Whether actin refers to the monomer or to the polymer should be clear from the context.

Domain swapping describes the phenomenon in which a heterodomain protein extends (part of) one of its domains to replace its counterpart in another protein. This can lead to formation of a homodimer, a homo-oligomer, or a hetero-oligomer (Liu and Eisenberg, 2002; Juy et al., 2003). Domain swapping can be extended to form a polymer. The term may refer to part of the functional cycle of a protein(s) or be invoked in the context of a long timescale to explain the evolution of oligomeric proteins (Hakansson and Linse, 2002; Rousseau and Schymkowitz, 2003).

Many proteins are enzymes; they function as catalysts and are classified with a four number Enzyme Classification (EC; 1992) code. Often one EC code number corresponds to one homolog family; however, there are many exceptions. Proteins in one homolog family may have different substrates or mechanisms and hence several codes; conversely, one catalytic function may be performed by more than one (family of) protein(s). The binding of substrate and subsequent catalysis may occur within a single domain or at the interface of two domains or even subunits; other domains or subunits may be involved in regulation. Different isoforms of the protein may have different other domains spliced to it (Fig. 1-3).

FIGURE 1-3 Domains of 3-deoxy-D-arabinoheptulosonic acid synthase (DAHPS). The middle panel shows the backbone of KDOPSEc (3-deoxy-D-manno-octulosonate-8-phosphate synthase) from Eschericia coli, a member of the (β/α)8 homolog family. The core consists of eight parallel βstrands (blue), arranged sequentially to form a barrel; they are connected by eight α helices (yellow). To the left is DAHPSEc (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase), a paralog of KDOPSEc, that has the same (β/α)8 core. It contains two extra elements of secondary structure, βstrands β6a and β6b, that provide the binding site for the feedback inhibitors—phenylalanine, tyrosine, or tryptophan (specific to the appropriate isoform). To the right is DAHPSTm from Thermotoga maritima. It lacks the β6a and β6b loop of DAHPSEc, but instead has acquired by gene splicing and fusion a flavodoxin like (FL) domain. It is also subject to feedback regulation by tyrosine and tryptophan, which bind to the FL domain. However, the pathway and mechanism of inhibition is entirely different in the two orthologs (Shumilin et al.,