analyses of biological macromolecules
Biological macromolecules are polar
The main point of the first segment of this material is this: THE MONOMER UNITS OF BIOLOGICAL MACROMOLECULES HAVE HEADS AND TAILS. WHEN THEY POLYMERIZE IN A HEAD-TO-TAIL FASHION, THE RESULTING POLYMERS ALSO HAVE HEADS AND TAILS.
These macromolecules are polar [polar: having different ends] because they are formed by head to tail condensation of polar monomers. Let's look at the three major classes of macromolecules to see how this works, and let's begin with carbohydrates.
Monosaccharides polymerize to yield polysaccharides.
Glucose is a typical monosaccharide. It has two important types of functional group: a carbonyl group (an aldehyde in glucose, some other sugars have a ketone group instead.) hydroxyl groups on the other carbons. This is what you need to know about glucose, not its detailed structure.
Glucose exists mostly in ring structures. ( 5-OH adds across the carbonyl oxygen double bond.) This is a so-called internal hemiacetal. The ring can close in either of two ways, giving rise to anomeric forms, -OH down (the alpha-form) and -OH up (the beta-form)
The anomeric carbon (the carbon to which this -OH is attached) differs significantly from the other carbons. (note: it's easy to pick out because it is the only carbon with TWO oxygens -- ring and hydroxyl -- attached.)
Free anomeric carbons have the chemical reactivity of carbonyl carbons because they spend part of their time in the open chain form. They can reduce alkaline solutions of cupric salts. Sugars with free anomeric carbons are therefore called reducing sugars. The rest of the carbohydrate consists of ordinary carbons and ordinary -OH groups. The point is, a monosaccharide can therefore be thought of as having polarity, with one end consisting of the anomeric carbon, and the other end consisting of the rest of the molecule.
Monosaccharides can polymerize by elimination of the elements of water between the anomeric hydroxyl and a hydroxyl of another sugar. This is called a glycosidic bond.
If two anomeric hydroxyl groups react (head to head condensation) the product has no reducing end (no free anomeric carbon). This is the case with sucrose
If the anomeric hydroxyl reacts with a non-anomeric hydroxyl of another sugar, the product has ends with different properties. A reducing end (with a free anomeric carbon). A nonreducing end. This is the case with maltose.
Since most monosaccharides have more than one hydroxyl, branches are possible, and are common. Branches result in a more compact molecule. If the branch ends are the reactive sites, more branches provide more reactive sites per molecule.
Let's now turn to nucleotides and nucleic acids.
Nucleotides polymerize to yield nucleic acids.
Nucleotides consist of three parts.
Phosphate.
Monosaccharide.
Ribose (in ribonucleotides)
Deoxyribose, which lacks a 2' -OH (in deoxyribonucleotides)
The presence or absence of the 2' -OH has structural significance that will be discussed later.
There are four dominant bases; here are three of them: adenine (purine) cytosine (pyrimidine) guanine (purine)
The fourth base is (a pyrimidine) uracil (in ribonucleotides) or thymine (in deoxyribonucleotides)
Be aware that uracil and thymine are very similar; they differ only by a methyl group. You need to know which are purines and which are pyrimidines, and whether it is the purines or the pyrimidines that have one ring. The reasons for knowing these points relate to the way purines and pyrimidines interact in nucleic acids, which we'll cover shortly.
Nucleotides polymerize by eliminating the elements of water to form esters between the 5'-phosphate and the 3' -OH of another nucleotide.
A 3'->5' phosphodiester bond is thereby formed. The product has ends with different properties. An end with a free 5' group (likely with phosphate attached); this is called the 5' end. An end with a free 3' group; this is called the 3' end.
Let's look at the conventions for writing sequences of nucleotides in nucleic acids. Bases are abbreviated by their initials: A, C, G and U or T. U is normally found only in RNA, and T is normally found only in DNA. So the presence of U vs. T distinguishes between RNA and DNA in a written sequence.
Sequences are written with the 5' end to the left and the 3' end to the right unless specifically designated otherwise.
Phosphate groups are usually not shown unless the writer wants to draw attention to them. The following representations are all equivalent. uracil adenine cytosine guanine | | | | P-ribose-P-ribose-P-ribose-P-ribose-OH 5' 3' 5' 3' 5' 3' 5' 3' pUpApCpG UACG 3' GCAU 5'
(Note that in the last line the sequence is written in reverse order , but the ends are appropriately designated.)
Branches are possible in RNA but not in DNA. RNA has a 2' -OH, at which branching could occur, while DNA does not. Branching is very unusual; it is known to occur only during RNA modification [the "lariat"], but not in any finished RNA species. Amino acids polymerize to form polypeptides or proteins. Amino acids contain a carboxylic acid (-COOH) group and an amino (-NH2) group. The amino groups are usually attached to the carbons which are alpha to the carboxyl carbons, so they are called alpha-amino acids.
The naturally occurring amino acids are optically active, as they have four different groups attached to one carbon, (Glycine is an exception, having two hydrogens) and have the L-configuration.
The R-groups of the amino acids provide a basis for classifying amino acids. There are many ways of classifying amino acids, but one very useful way is on the basis of how well or poorly the R-group interacts with water
The first class is the hydrophobic R-groups which can be aliphatic (such as the methyl group of alanine) or aromatic (such as the phenyl group of phenylalanine). The second class is the hydrophilic R-groups which can contain neutral polar (such as the -OH of serine) or ionizable (such as the -COOH of aspartate) functional groups.
I was born in Aarberg, Switzerland, on October 4, 1938, and during my childhood I lived in the small town of Lyss in the Berner Seeland. At the time this was a rural area of farmland, forests and rivers. The roots of the Wüthrich family are in an even more rural, mountainous area, the farming village of Trub in the Emmental near Bern. My mother's family owned the Restaurant "Bären" and a bakery in Lyss. My grandfather, Otto Kuchen, enjoyed fishing and hunting, and his jugged hare dish was a widely known fall season delicacy at the "Bären". My interests during childhood were largely influenced by our living in an old farmhouse, where my second grandfather, Jakob Wüthrich, had been a farmer. Although my father, Herrmann Wüthrich, took up an occupation as an accountant, he remained very much attached to his upbringings and our family produced a wide range of farming goods. My mother, Gertrud Wüthrich-Kuchen was the true center of our family life. In addition to raising me and my two younger sisters, Elisabeth and Ruth, she did marvelous things in the kitchen, tended our big garden, raised fowl, and was involved in various activities in the community.
My intense contacts with the rural environment of plants and animals awakened my interest in natural science at an early age. In particular, I acquired a thorough knowledge of the behavior of all sorts of water animals, mostly through observations made while enjoying all aspects of work and fun with a private trout river. On rare occasions I still enjoy fishing trips, and I am a member of the Mercury Bay Game Fishing Club in Whitianga, New Zealand, which lists Ernest Hemingway and Zane Grey among its all-time membership. With regard to my professional life, I had set my mind on becoming a forest engineer. Although I subsequently changed my mind in this regard, I still enjoy tending the family forest, which now contains trees that were planted by three generations of our family starting with my grandfather.
At the Mercury Bay Game Fishing Club in Whitianga, New Zealand, 1987.
My formal training toward an academic profession started in 1952, when I transferred from the village schools in Lyss to the Gymnasium in the nearby "bilingue" city of Biel/Bienne. During the Gymnasium years my interests widened beyond forestry and fishing. We had the good fortune that our science and language teachers were either former University professors, who had left their academic positions elsewhere in Europe during the Second World War and found a haven in Biel, or followed the then common practice of using a teaching assignment at Gymnasium level as a stepping-stone for an academic career. At age 14 to 18 we were a group of seven students specializing in "natural sciences" who were thus trained in mathematics and physics at university level, and I happily accepted the challenge. According to my mother, it was during those years that I got used to working through the nights. Another focus was the French language, French literature, and French theatre and movies, which was largely motivated by the fact that the composition of our class as well as our teachers represented the bilingual character of Biel/Bienne. The Gymnasium Biel was informally attached to the Swiss Federal School of Sports and Gymnastics in nearby Magglingen, and thus my interest in competitive sports was awakened. These three areas all play an important role in my life up to the present days. Physics and mathematics are key activities in my professional life, professional visits in Paris and "les provinces" are combined with the sampling of French food, wine and culture, and I not only obtained the "Eidgenössisches Turn- und Sportlehrerdiplom" as one of my University degrees, but also played in a competitive soccer league well beyond the age of 50.
As a postdoctoral student in Berkeley, 1966.
By now I can look back on 40 years of intense involvement with techniques referred to as "magnetic resonance spectroscopy". At the outset in 1962 and throughout my graduate studies there was electron paramagnetic resonance (EPR spectroscopy). EPR was complemented during my postdoctoral training from 1965-1967 by nuclear magnetic resonance (NMR) spectroscopy applied to chemical physics projects, and since the fall of 1967 I have used NMR for studies of biological macromolecules. From there it was a sinuous avenue that led by 1984 to the NMR method for protein structure determination in solution. Our results were occasionally met with doubts and disbelief, so that considerable moral strength and perseverance was at times called for.
During my student years from 1957-1962, NMR spectroscopy was just being introduced as an analytical tool in chemistry, molecular biology was not yet established as an independent discipline, and the initial three-dimensional protein crystal structures were just emerging. My education at the University of Bern could thus not possibly cover the areas of our current research. The faculty and the student classes in Bern were small in numbers, with three physics students and seven chemistry students starting in 1957. From my curriculum in chemistry, physics and mathematics, I best remember intense work in linear algebra, classical mechanics, chemical thermodynamics, physical chemistry of synthetic polymers, and preparative biochemistry of proteins and nucleic acids. This combination turned out to be an excellent foundation for my later scientific activities. The last two years of formal education, from 1962 to 1964, were spent at the University of Basel, majoring in sports and getting a Ph.D. in chemistry. Studying sports included about 25 weekly hours of intense physical exercise as well as premedical courses in human anatomy and physiology. Combined with experience gained from observations made on myself in the pursuit of competitive sports, this provided an additional dimension to my education. The subject of my Ph.D. thesis in inorganic chemistry with Professor Silvio Fallab was the catalytic activity of copper compounds in autoxidation reactions, and for this project the availability of a state-of-the-art EPR spectrometer in the Physics Institute was a great opportunity.
Studying natural sciences has always been a lot of fun for me, but nonetheless my mind was quite solidly set on a career as a high school teacher with a heavy involvement in sports. In parallel to my studies in natural sciences, I extensively yielded to what I thought to be my vocation. Thus, during the years 1957-1962, I spent part of each winter as a ski instructor in Swiss mountain resorts. From 1959 to 1965, I had part-time jobs in high schools, first teaching physics at the Kantonsschule Solothurn, then chemistry at the Gymnasium Biel, and finally gymnastics at the Mädchengymnasium in Basel. These teaching assignments also had an important impact on my personal life. In 1961, while on my job as a ski instructor in the resort town of Saanenmöser in the Berner Oberland, I met my wife, Marianne Briner, who at the time was an elementary school teacher. We were married in 1963, and Marianne then joined me in studying sports at the University of Basel, graduating with the "Eidgenössisches Turn- und Sportlehrerdiplom" and specializing in modern dance. After the graduate student and postdoctoral years we started a family, with our son Bernhard Andrew being born in 1968 in Berkeley Heights, NJ, USA, and our daughter Karin Lynn joining us in 1970 in Greifensee near Zürich, Switzerland.
After finishing my graduate studies I spent another year in Basel concentrating on EPR studies of metal complexes in solution. In the spring of 1965 we moved to the USA, where I joined Professor Robert E. Connick at the University of California, Berkeley, for postdoctoral training. We used NMR spin relaxation measurements of 17O, 2H and 1H in addition to EPR for studies of the hydration of metal ions and metal complexes. The Berkeley period was devoted to intensive work on the theory of nuclear spin relaxation, group theory and quantum mechanics, which was motivated by Bob Connick's weekly group seminar, a graduate course on "Group Theory and Quantum Mechanics" by Professor Michael Tinkham, and an intense collaboration with another Swiss postdoc, Alex von Zelewsky, who soon thereafter accepted the chair of inorganic chemistry at the University of Fribourg in Switzerland. Over the years, Marianne and I returned at regular intervals to Berkeley, to renew the friendships of the 1960s and revive fond memories.
In October 1967 I joined the Biophysics Department of Dr. Robert G. Shulman at Bell Telephone Laboratories in Murray Hill, New Jersey. I was given responsibility for the maintenance of what was one of the first superconducting high resolution NMR spectrometers, which operated at a proton resonance frequency of 220 MHz, and I was otherwise free to use this instrument for "research on protein structure and function". Due to my background, my interest was focused on metal centers rather than on polypeptide chains, and all my initial projects in high resolution NMR had to do with hemoproteins. Using blood sampled from my arm in the first aid station, a Japanese colleague at Bell Labs, Dr. Tetsuo Yamane, prepared "hemoglobin (KW)", and within a few months we found entirely new avenues of deriving information on structure-function correlations from the NMR spectra of hemoglobin and other hemoproteins. These projects were a lucky choice: with the limited sensitivity and spectral resolution of the instrumentation available in 1968, the special spectral properties of hemoproteins were a great asset for successful NMR applications. Many years later, the unique NMR spectral features that enabled the early work with these metalloproteins had an important role in various aspects of the development of the NMR method for threedimensional protein structure determination.
In October 1969 I returned to Switzerland to join the ETH Zürich. From the start I was equally well equipped with NMR and EPR instrumentation as previously at Bell Telephone Laboratories, and during the following 32 years the ETH provided us in regular intervals with the most advanced NMR equipment. Until 1975 I was working with a small group of students, a chemical engineer, Rudolf Baumann, who has stayed with me throughout all these years, and a postdoctoral associate with a physics Ph.D. in solid state EPR, Dr. Regula Keller, who largely took responsibility for the research with hemoproteins from 1970 to 1982. In 1973, Gerhard Wagner decided to do his graduate work with me. Gerhard then stayed with the group until 1987, pursuing a classical European academic career with Habilitation before settling as a Professor at Harvard Medical School. Being able to keep outstanding junior scientists as research associates over extended periods of time was a special privilege enjoyed by senior faculty in the traditional "European system", and the continued presence of Rudolf, Regula and Gerhard during most of my initial 15 years in Zürich was a key factor for success with our research program.
In Zürich, we continued research on hemoproteins with the use of NMR and EPR spectroscopy, where the biochemical work was mostly done by groups outside of the ETH who joined us for collaborative projects, and the spectroscopic work was done by Regula Keller, myself and a succession of graduate students. In addition, we started a program of systematic studies on the application of NMR techniques with polypeptides and small proteins. Spirits were kept high by successful studies of cyclic peptides in collaboration with the Head of the Institute of Molecular Biology and Biophysics, Professor Robert Schwyzer, the observation of unexpectedly well-resolved and longlived NMR lines of amide protons in the small protein basic pancreatic trypsin inhibitor (BPTI), and the discovery of "ring flips" in BPTI. On the main line of research, which should lead to a method for protein structure determination in solution, there was only little progress. In 1975, in an attempt to survey the state of the field of NMR spectroscopy with biological macromolecules, I wrote the monograph NMR in Biological Research: Peptides and Proteins. There were two principal conclusions from this venture that should greatly affect the continuation of our work plan. First, I fully realized that we really had been extremely fortunate in choosing hemoproteins as a focus for our early NMR efforts. Second, it became clear that attempts of the early 1970s to derive de novo three-dimensional protein structures from conformation-dependent proton chemical shifts was not a promising approach, independent of whether these shifts were caused by intrinsic or extrinsic diamagnetic or paramagnetic probes. We thus had to look for novel avenues for NMR structure determination, where hemoproteins with their unique NMRspectral properties could be an ideal testing ground for new ideas.